Engineered glucosyltransferases

ABSTRACT

Disclosed herein are glucosyltransferases with modified amino acid sequences. Such engineered enzymes synthesize insoluble alpha-glucan products having decreased molecular weight. Further disclosed are reactions and methods in which engineered glucosyltransferases are used to produce insoluble alpha-glucan.

This application claims the benefit of U.S. Provisional Application Nos.62/640,708 (filed Mar. 9, 2018) and 62/792,449 (filed Jan. 15, 2019),which are incorporated herein by reference in their entirety.

FIELD

The present disclosure is in the field of enzyme catalysis. For example,the disclosure pertains to glucosyltransferase enzymes with modifiedamino acid sequences. Such modified enzymes synthesize products withdecreased molecular weight.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The official copy of the sequence listing is submitted electronicallyvia EFS-Web as an ASCII formatted sequence listing with a file named20190306_CL6607USNP_SequenceListing.txt, created on Mar. 6, 2019, andhaving a size of about 315 kilobytes and is filed concurrently with thespecification. The sequence listing contained in this ASCII-formatteddocument is part of the specification and is herein incorporated byreference in its entirety.

BACKGROUND

Driven by a desire to use polysaccharides in various applications,researchers have explored for polysaccharides that are biodegradable andthat can be made economically from renewably sourced feedstocks. Onesuch polysaccharide is alpha-1,3-glucan, an insoluble glucan polymercharacterized by having alpha-1,3-glycosidic linkages. This polymer hasbeen prepared, for example, using a glucosyltransferase enzyme isolatedfrom Streptococcus salivarius (Simpson et al., Microbiology141:1451-1460, 1995). Also for example, U.S. Pat. No. 7,000,000disclosed the preparation of a spun fiber from enzymatically producedalpha-1,3-glucan. Various other glucan materials have also been studiedfor developing new or enhanced applications. For example, U.S. PatentAppl. Publ. No. 2015/0232819 discloses enzymatic synthesis of severalinsoluble glucans having mixed alpha-1,3 and -1,6 linkages.

While these and other advances have been made in producing insolubleglucan polymers using glucosyltransferase enzymes, less attentionappears to have been drawn to modulating the molecular weight ofinsoluble glucan products synthesized by such enzymes. Addressing thistechnological gap, disclosed herein are glucosyltransferases withmodified amino acid sequences that produce lower molecular weightinsoluble glucan products.

SUMMARY

In one embodiment, the present disclosure concerns a non-nativeglucosyltransferase comprising at least one amino acid substitution at aposition corresponding with amino acid residue Leu-513, Pro-550,Ser-553, Asn-557, Asn-558, Trp-571, Asn-573, Asp-575, Lys-578, Asn-581,Thr-585, or Gln-616 of SEQ ID NO:62, wherein the non-nativeglucosyltransferase synthesizes insoluble alpha-glucan comprising1,3-glycosidic linkages, and the molecular weight of the insolublealpha-glucan is lower than the molecular weight of insolublealpha-glucan synthesized by a second glucosyltransferase that onlydiffers from the non-native glucosyltransferase at the substitutionposition(s).

In another embodiment, the present disclosure concerns a polynucleotidecomprising a nucleotide sequence encoding a non-nativeglucosyltransferase as disclosed herein, optionally wherein one or moreregulatory sequences are operably linked to the nucleotide sequence, andpreferably wherein the one or more regulatory sequences include apromoter sequence.

In another embodiment, the present disclosure concerns a reactioncomposition comprising water, sucrose, and a non-nativeglucosyltransferase as disclosed herein.

In another embodiment, the present disclosure concerns a method ofproducing insoluble alpha-glucan comprising 1,3-glycosidic linkages, themethod comprising: (a) contacting at least water, sucrose, and anon-native glucosyltransferase enzyme as disclosed herein, wherebyinsoluble alpha-glucan comprising 1,3-glycosidic linkages is produced;and (b) optionally, isolating the insoluble alpha-glucan produced instep (a).

In another embodiment, the present disclosure concerns a method ofpreparing a polynucleotide sequence encoding a non-nativeglucosyltransferase as disclosed herein, the method comprising: (a)identifying a polynucleotide sequence encoding a parentglucosyltransferase that (i) comprises an amino acid sequence that is atleast about 40% identical to SEQ ID NO:4 or SEQ ID NO:66 (positions55-960 of SEQ ID NO:4), and (ii) synthesizes insoluble alpha-glucancomprising 1,3-glycosidic linkages; and (b) modifying the polynucleotidesequence identified in step (a) to substitute at least one amino acid ofthe parent glucosyltransferase at a position corresponding with aminoacid residue Leu-513, Pro-550, Ser-553, Asn-557, Asn-558, Trp-571,Asn-573, Asp-575, Lys-578, Asn-581, Thr-585, or Gln-616 of SEQ ID NO:62,thereby providing a polynucleotide sequence encoding a non-nativeglucosyltransferase that synthesizes insoluble alpha-glucan comprising1,3-glycosidic linkages, wherein the molecular weight of the insolublealpha-glucan is lower than the molecular weight of insolublealpha-glucan synthesized by the parent glucosyltransferase.

BRIEF DESCRIPTION OF THE SEQUENCES

TABLE 1 Summary of Nucleic Acid and Protein SEQ ID Numbers^(b) Nucleicacid Protein Description SEQ ID NO. SEQ ID NO. GTF 0874, Streptococcussobrinus. The first 156 amino acids  1^(a) 2 of the protein are deletedcompared to GENBANK (1435 aa) Identification No. 450874; a startmethionine is included. GTF 6855, Streptococcus salivarius SK126. Thefirst 178  3^(a) 4 amino acids of the protein are deleted compared to(1341 aa) GENBANK Identification No. 228476855 (Acc. No. ZP_04061500.1);a start methionine is included. GTF 2379, Streptococcus salivarius. Thefirst 203 amino  5^(a) 6 acids of the protein are deleted compared toGENBANK (1247 aa) Identification No. 662379; a start methionine isincluded. GTF 7527 or GTFJ, Streptococcus salivarius. The first 42 7^(a) 8 amino acids of the protein are deleted compared to (1477 aa)GENBANK Identification No. 47527; a start methionine is included. GTF1724, Streptococcus downei. The first 162 amino acids  9^(a) 10 of theprotein are deleted compared to GENBANK (1436 aa) Identification No.121724; a start methionine is included. GTF 0544, Streptococcus mutans.The first 164 amino acids 11^(a) 12 of the protein are deleted comparedto GENBANK (1313 aa) Identification No. 290580544; a start methionine isincluded. GTF 5926, Streptococcus dentirousetti. The first 144 amino13^(a) 14 acids of the protein are deleted compared to GENBANK (1323 aa)Identification No. 167735926; a start methionine is included. GTF 4297,Streptococcus oralis. The first 228 amino acids of 15^(a) 16 the proteinare deleted compared to GENBANK Identification (1348 aa) No. 7684297; astart methionine is included. GTF 5618, Streptococcus sanguinis. Thefirst 223 amino 17^(a) 18 acids of the protein are deleted compared toGENBANK (1348 aa) Identification No. 328945618; a start methionine isincluded. GTF 2765, unknown Streptococcus sp. C150. The first 193 19^(a)20 amino acids of the protein are deleted compared to (1340 aa) GENBANKIdentification No. 322372765; a start methionine is included. GTF 0427,Streptococcus sobrinus. The first 156 amino acids 25^(a) 26 of theprotein are deleted compared to GENBANK (1435 aa) Identification No.940427; a start methionine is included. GTF 2919, Streptococcussalivarius PS4. The first 92 amino 27^(a) 28 acids of the protein aredeleted compared to GENBANK (1340 aa) Identification No. 383282919; astart methionine is included. GTF 2678, Streptococcus salivarius K12.The first 188 amino 29^(a) 30 acids of the protein are deleted comparedto GENBANK (1341 aa) Identification No. 400182678; a start methionine isincluded. GTF 3929, Streptococcus salivarius JIM8777. The first 17833^(a) 34 amino acids of the protein are deleted compared to (1341 aa)GENBANK Identification No. 387783929; a start methionine is included.GTF 3298, Streptococcus sp. C150. The first 209 amino 59 acids of theprotein are deleted compared to GENBANK (1242 aa) Identification No.322373298; a start methionine is included. Wild type GTFJ, Streptococcussalivarius. GENBANK 60 Identification No. 47527. (1518 aa) Wild type GTFcorresponding to GTF 2678, Streptococcus 61 salivarius K12. (1528 aa)Wild type GTF corresponding to GTF 6855, Streptococcus 62 salivariusSK126. (1518 aa) Wild type GTF corresponding to GTF 2919, Streptococcus63 salivarius PS4. (1431 aa) Wild type GTF corresponding to GTF 2765,unknown 64 Streptococcus sp. C150. (1532 aa) Shorter version of GTF7527, Streptococcus salivarius, (also 65 referred to as “7527-NT”herein. The first 178 amino acids of (1341 aa) the protein are deletedcompared to GENBANK Identification No. 47527; a start methionine isincluded. ^(a)This DNA coding sequence is codon-optimized for expressionin E. coli, and is merely disclosed as an example of a suitable codingsequence. ^(b)SEQ ID NOs: 21-24, 31, 32 and 35-58 are intentionally notincluded in this table and merely serve as placeholders.

DETAILED DESCRIPTION

The disclosures of all cited patent and non-patent literature areincorporated herein by reference in their entirety.

Unless otherwise disclosed, the terms “a” and “an” as used herein areintended to encompass one or more (i.e., at least one) of a referencedfeature.

Where present, all ranges are inclusive and combinable, except asotherwise noted. For example, when a range of “1 to 5” (i.e., 1-5) isrecited, the recited range should be construed as including ranges “1 to4”, “1 to 3”, “1-2”, “1-2 & 4-5”, “1-3 & 5”, and the like.

The terms “alpha-glucan”, “alpha-glucan polymer” and the like are usedinterchangeably herein. An alpha-glucan is a polymer comprising glucosemonomeric units linked together by alpha-glycosidic linkages. In typicalembodiments, an alpha-glucan herein comprises 100% alpha-glycosidiclinkages, or at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%alpha-glycosidic linkages. Examples of alpha-glucan polymers hereininclude alpha-1,3-glucan.

The terms “poly alpha-1,3-glucan”, “alpha-1,3-glucan”, “alpha-1,3-glucanpolymer” and the like are used interchangeably herein. Alpha-1,3-glucanis a polymer comprising glucose monomeric units linked together byglycosidic linkages, wherein at least about 50% of the glycosidiclinkages are alpha-1,3. Alpha-1,3-glucan in certain embodimentscomprises at least 90% or 95% alpha-1,3 glycosidic linkages. Most or allof the other linkages in alpha-1,3-glucan herein typically arealpha-1,6, though some linkages may also be alpha-1,2 and/or alpha-1,4.

The terms “glycosidic linkage”, “glycosidic bond”, “linkage” and thelike are used interchangeably herein and refer to the covalent bondsconnecting the sugar monomers within a saccharide compound(oligosaccharides and/or polysaccharides). The term“alpha-1,3-glycosidic linkage” as used herein refers to the type ofcovalent bond that joins alpha-D-glucose molecules to each other throughcarbons 1 and 3 on adjacent alpha-D-glucose rings. The term“alpha-1,6-glycosidic linkage” as used herein refers to the covalentbond that joins alpha-D-glucose molecules to each other through carbons1 and 6 on adjacent alpha-D-glucose rings. The glycosidic linkages of aglucan polymer herein can also be referred to as “glucosidic linkages”.Herein, “alpha-D-glucose” will be referred to as “glucose”.

The glycosidic linkage profile of an alpha-glucan herein can bedetermined using any method known in the art. For example, a linkageprofile can be determined using methods using nuclear magnetic resonance(NMR) spectroscopy (e.g., ¹³C NMR and/or ¹H NMR). These and othermethods that can be used are disclosed in, for example, FoodCarbohydrates: Chemistry, Physical Properties, and Applications (S. W.Cui, Ed., Chapter 3, S. W. Cui, Structural Analysis of Polysaccharides,Taylor & Francis Group LLC, Boca Raton, Fla., 2005), which isincorporated herein by reference.

The “molecular weight” of large alpha-glucan polymers herein can berepresented as weight-average molecular weight (Mw) or number-averagemolecular weight (Mn), the units of which are in Daltons or grams/mole.Alternatively, the molecular weight of large alpha-glucan polymers canbe represented as DPw (weight average degree of polymerization) or DPn(number average degree of polymerization). The molecular weight ofsmaller alpha-glucan polymers such as oligosaccharides typically can beprovided as “DP” (degree of polymerization), which simply refers to thenumber of glucoses comprised within the alpha-glucan; “DP” can alsocharacterize the molecular weight of a polymer on an individual moleculebasis. Various means are known in the art for calculating these variousmolecular weight measurements such as with high-pressure liquidchromatography (HPLC), size exclusion chromatography (SEC), or gelpermeation chromatography (GPC).

The term “sucrose” herein refers to a non-reducing disaccharide composedof an alpha-D-glucose molecule and a beta-D-fructose molecule linked byan alpha-1,2-glycosidic bond. Sucrose is known commonly as table sugar.Sucrose can alternatively be referred to as“alpha-D-glucopyranosyl-(1→2)-beta-D-fructofuranoside”.“Alpha-D-glucopyranosyl” and “glucosyl” are used interchangeably herein.

The terms “glucosyltransferase”, “glucosyltransferase enzyme”, “GTF”,“glucansucrase” and the like are used interchangeably herein. Theactivity of a glucosyltransferase herein catalyzes the reaction of thesubstrate sucrose to make the products alpha-glucan and fructose. Otherproducts (by-products) of a GTF reaction can include glucose, varioussoluble gluco-oligosaccharides, and leucrose. Wild type forms ofglucosyltransferase enzymes generally contain (in the N-terminal toC-terminal direction) a signal peptide (which is typically removed bycleavage processes), a variable domain, a catalytic domain, and aglucan-binding domain. A glucosyltransferase herein is classified underthe glycoside hydrolase family 70 (GH70) according to the CAZy(Carbohydrate-Active EnZymes) database (Cantarel et al., Nucleic AcidsRes. 37:D233-238, 2009).

The term “glucosyltransferase catalytic domain” herein refers to thedomain of a glucosyltransferase enzyme that providesalpha-glucan-synthesizing activity to a glucosyltransferase enzyme. Aglucosyltransferase catalytic domain typically does not require thepresence of any other domains to have this activity.

The terms “enzymatic reaction”, “glucosyltransferase reaction”, “glucansynthesis reaction”, “reaction composition”, “reaction formulation” andthe like are used interchangeably herein and generally refer to areaction that initially comprises water, sucrose, at least one activeglucosyltransferase enzyme, and optionally other components. Componentsthat can be further present in a glucosyltransferase reaction typicallyafter it has commenced include fructose, glucose, leucrose, solublegluco-oligosaccharides (e.g., DP2-DP7) (such may be considered asproducts or by-products, depending on the glucosyltransferase used),and/or insoluble alpha-glucan product(s) of DP8 or higher (e.g., DP100and higher). It would be understood that certain glucan products, suchas alpha-1,3-glucan with a degree of polymerization (DP) of at least 8or 9, are water-insoluble and thus not dissolved in a glucan synthesisreaction, but rather may be present out of solution (e.g., by virtue ofhaving precipitated from the reaction). It is in a glucan synthesisreaction where the step of contacting water, sucrose and aglucosyltransferase enzyme is performed. The term “under suitablereaction conditions” as used herein refers to reaction conditions thatsupport conversion of sucrose to alpha-glucan product(s) viaglucosyltransferase enzyme activity.

The terms “percent by volume”, “volume percent”, “vol %”, “v/v %” andthe like are used interchangeably herein. The percent by volume of asolute in a solution can be determined using the formula: [(volume ofsolute)/(volume of solution)]×100%.

The terms “percent by weight”, “weight percentage (wt %)”,“weight-weight percentage (% w/w)” and the like are used interchangeablyherein. Percent by weight refers to the percentage of a material on amass basis as it is comprised in a composition, mixture, or solution.

The terms “aqueous conditions”, “aqueous reaction conditions”, “aqueoussetting”, “aqueous system” and the like are used interchangeably herein.Aqueous conditions herein refer to a solution or mixture in which thesolvent is at least about 60 wt % water, for example. Aglucosyltransferase reaction herein is performed under aqueousconditions.

A glucan that is “insoluble”, “aqueous-insoluble”, “water-insoluble”(and like terms) (e.g., alpha-1,3-glucan with a DP of 8 or higher) doesnot dissolve (or does not appreciably dissolve) in water or otheraqueous conditions, optionally where the aqueous conditions are furthercharacterized to have a pH of 4-9 (e.g., pH 6-8) and/or temperature ofabout 1 to 85° C. (e.g., 20-25° C.). In contrast, glucans such ascertain oligosaccharides herein that are “soluble”, “aqueous-soluble”,“water-soluble” and the like (e.g., alpha-1,3-glucan with a DP less than8) appreciably dissolve under these conditions.

The terms “polynucleotide”, “polynucleotide sequence”, “nucleic acidmolecule” and the like are used interchangeably herein. These termsencompass nucleotide sequences and the like. A polynucleotide may be apolymer of DNA or RNA that is single- or double-stranded, thatoptionally contains synthetic, non-natural or altered nucleotide bases.A polynucleotide may be comprised of one or more segments of cDNA,genomic DNA, synthetic DNA, or mixtures thereof.

The term “gene” as used herein refers to a DNA polynucleotide sequencethat expresses an RNA (RNA is transcribed from the DNA polynucleotidesequence) from a coding region, which RNA can be a messenger RNA(encoding a protein) or a non-protein-coding RNA. A gene may refer tothe coding region alone, or may include regulatory sequences upstreamand/or downstream to the coding region (e.g., promoters, 5′-untranslatedregions, 3′-transcription terminator regions). A coding region encodinga protein can alternatively be referred to herein as an “open readingframe” (ORF). A gene that is “native” or “endogenous” refers to a geneas found in nature with its own regulatory sequences; such a gene islocated in its natural location in the genome of a host cell. A“chimeric” gene refers to any gene that is not a native gene, comprisingregulatory and coding sequences that are not found together in nature(i.e., the regulatory and coding regions are heterologous with eachother). Accordingly, a chimeric gene may comprise regulatory sequencesand coding sequences that are derived from different sources, orregulatory sequences and coding sequences derived from the same source,but arranged in a manner different than that found in nature. A“foreign” or “heterologous” gene can refer to a gene that is introducedinto the host organism by gene transfer. Foreign/heterologous genes cancomprise native genes inserted into a non-native organism, native genesintroduced into a new location within the native host, or chimericgenes. Polynucleotide sequences in certain embodiments disclosed hereinare heterologous. A “transgene” is a gene that has been introduced intothe genome by a gene delivery procedure (e.g., transformation). A“codon-optimized” open reading frame has its frequency of codon usagedesigned to mimic the frequency of preferred codon usage of the hostcell.

As used herein, the term “polypeptide” is defined as a chain of aminoacid residues, usually having a defined sequence. As used herein theterm polypeptide is interchangeable with the terms “peptides” and“proteins”. Typical amino acids contained in polypeptides herein include(respective three- and one-letter codes shown parenthetically): alanine(Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp,D), cysteine (Cys, C), glutamic acid (Glu, E), glutamine (Gln, Q),glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu,L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F),proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp,W), tyrosine (Tyr, Y), valine (Val, V).

The term “heterologous” means not naturally found in the location ofinterest. For example, a heterologous gene can be one that is notnaturally found in a host organism, but that is introduced into the hostorganism by gene transfer. As another example, a nucleic acid moleculethat is present in a chimeric gene can be characterized as beingheterologous, as such a nucleic acid molecule is not naturallyassociated with the other segments of the chimeric gene (e.g., apromoter can be heterologous to a coding sequence).

A “non-native” amino acid sequence or polynucleotide sequence comprisedin a cell or organism herein does not occur in a native (natural)counterpart of such cell or organism. Such an amino acid sequence orpolynucleotide sequence can also be referred to as being heterologous tothe cell or organism.

“Regulatory sequences” as used herein refer to nucleotide sequenceslocated upstream of a gene's transcription start site (e.g., promoter),5′ untranslated regions, introns, and 3′ non-coding regions, and whichmay influence the transcription, processing or stability, and/ortranslation of an RNA transcribed from the gene. Regulatory sequencesherein may include promoters, enhancers, silencers, 5′ untranslatedleader sequences, introns, polyadenylation recognition sequences, RNAprocessing sites, effector binding sites, stem-loop structures, andother elements involved in regulation of gene expression. One or moreregulatory elements herein may be heterologous to a coding regionherein.

A “promoter” as used herein refers to a DNA sequence capable ofcontrolling the transcription of RNA from a gene. In general, a promotersequence is upstream of the transcription start site of a gene.Promoters may be derived in their entirety from a native gene, or becomposed of different elements derived from different promoters found innature, or even comprise synthetic DNA segments. Promoters that cause agene to be expressed in a cell at most times under all circumstances arecommonly referred to as “constitutive promoters”. A promoter mayalternatively be inducible. One or more promoters herein may beheterologous to a coding region herein.

A “strong promoter” as used herein refers to a promoter that can directa relatively large number of productive initiations per unit time,and/or is a promoter driving a higher level of gene transcription thanthe average transcription level of the genes in a cell.

The terms “3′ non-coding sequence”, “transcription terminator”,“terminator” and the like as used herein refer to DNA sequences locateddownstream of a coding sequence. This includes polyadenylationrecognition sequences and other sequences encoding regulatory signalscapable of affecting mRNA processing or gene expression.

As used herein, a first nucleic acid sequence is “hybridizable” to asecond nucleic acid sequence when a single-stranded form of the firstnucleic acid sequence can anneal to the second nucleic acid sequenceunder suitable annealing conditions (e.g., temperature, solution ionicstrength). Hybridization and washing conditions are well known andexemplified in Sambrook J, Fritsch E F and Maniatis T, MolecularCloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory:Cold Spring Harbor, N.Y. (1989), which is incorporated herein byreference, particularly Chapter 11 and Table 11.1.

The term “DNA manipulation technique” refers to any technique in whichthe sequence of a DNA polynucleotide sequence is modified. Although theDNA polynucleotide sequence being modified can be used as a substrateitself for modification, it does not have to be physically in hand forcertain techniques (e.g., a sequence stored in a computer can be used asthe basis for the manipulation technique). A DNA manipulation techniquecan be used to delete and/or mutate one or more DNA sequences in alonger sequence. Examples of a DNA manipulation technique includerecombinant DNA techniques (restriction and ligation, molecularcloning), polymerase chain reaction (PCR), and synthetic DNA methods(e.g., oligonucleotide synthesis and ligation). Regarding synthetic DNAtechniques, a DNA manipulation technique can entail observing a DNApolynucleotide in silico, determining desired modifications (e.g., oneor more deletions) of the DNA polynucleotide, and synthesizing a DNApolynucleotide that contains the desired modifications.

The term “in silico” herein means in or on an information storage and/orprocessing device such as a computer; done or produced using computersoftware or simulation, i.e., virtual reality.

The terms “upstream” and “downstream” as used herein with respect topolynucleotides refer to “5′ of” and “3′ of”, respectively.

The term “expression” as used herein refers to (i) transcription of RNA(e.g., mRNA or a non-protein-coding RNA) from a coding region, and/or(ii) translation of a polypeptide from mRNA. Expression of a codingregion of a polynucleotide sequence can be up-regulated ordown-regulated in certain embodiments.

The term “operably linked” as used herein refers to the association oftwo or more nucleic acid sequences such that the function of one isaffected by the other. For example, a promoter is operably linked with acoding sequence when it is capable of affecting the expression of thatcoding sequence. That is, the coding sequence is under thetranscriptional control of the promoter. A coding sequence can beoperably linked to one (e.g., promoter) or more (e.g., promoter andterminator) regulatory sequences, for example.

The term “recombinant” when used herein to characterize a DNA sequencesuch as a plasmid, vector, or construct refers to an artificialcombination of two otherwise separated segments of sequence, e.g., bychemical synthesis and/or by manipulation of isolated segments ofnucleic acids by genetic engineering techniques.

The term “transformation” as used herein refers to the transfer of anucleic acid molecule into a host organism or host cell by any method. Anucleic acid molecule that has been transformed into an organism/cellmay be one that replicates autonomously in the organism/cell, or thatintegrates into the genome of the organism/cell, or that existstransiently in the cell without replicating or integrating. Non-limitingexamples of nucleic acid molecules suitable for transformation aredisclosed herein, such as plasmids and linear DNA molecules. Hostorganisms/cells herein containing a transforming nucleic acid sequencecan be referred to as “transgenic”, “recombinant”, “transformed”,“engineered”, as a “transformant”, and/or as being “modified forexogenous gene expression”, for example.

The terms “sequence identity”, “identity” and the like as used hereinwith respect to polynucleotide or polypeptide sequences refer to thenucleic acid residues or amino acid residues in two sequences that arethe same when aligned for maximum correspondence over a specifiedcomparison window. Thus, “percentage of sequence identity”, “percentidentity” and the like refer to the value determined by comparing twooptimally aligned sequences over a comparison window, wherein theportion of the polynucleotide or polypeptide sequence in the comparisonwindow may comprise additions or deletions (i.e., gaps) as compared tothe reference sequence (which does not comprise additions or deletions)for optimal alignment of the two sequences. The percentage is calculatedby determining the number of positions at which the identical nucleicacid base or amino acid residue occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the window of comparison andmultiplying the results by 100 to yield the percentage of sequenceidentity. It would be understood that, when calculating sequenceidentity between a DNA sequence and an RNA sequence, T residues of theDNA sequence align with, and can be considered “identical” with, Uresidues of the RNA sequence. For purposes of determining “percentcomplementarity” of first and second polynucleotides, one can obtainthis by determining (i) the percent identity between the firstpolynucleotide and the complement sequence of the second polynucleotide(or vice versa), for example, and/or (ii) the percentage of basesbetween the first and second polynucleotides that would create canonicalWatson and Crick base pairs.

Percent identity can be readily determined by any known method,including but not limited to those described in: 1) ComputationalMolecular Biology (Lesk, A. M., Ed.) Oxford University: NY (1988); 2)Biocomputing: Informatics and Genome Projects (Smith, D. W., Ed.)Academic: NY (1993); 3) Computer Analysis of Sequence Data, Part I(Griffin, A. M., and Griffin, H. G., Eds.) Humana: NJ (1994); 4)Sequence Analysis in Molecular Biology (von Heinje, G., Ed.) Academic(1987); and 5) Sequence Analysis Primer (Gribskov, M. and Devereux, J.,Eds.) Stockton: NY (1991), all of which are incorporated herein byreference.

Preferred methods for determining percent identity are designed to givethe best match between the sequences tested. Methods of determiningidentity and similarity are codified in publicly available computerprograms, for example. Sequence alignments and percent identitycalculations can be performed using the MEGALIGN program of theLASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.),for example. Multiple alignment of sequences can be performed, forexample, using the Clustal method of alignment which encompasses severalvarieties of the algorithm including the Clustal V method of alignment(described by Higgins and Sharp, CABIOS. 5:151-153 (1989); Higgins, D.G. et al., Comput. Appl. Biosci., 8:189-191 (1992)) and found in theMEGALIGN v8.0 program of the LASERGENE bioinformatics computing suite(DNASTAR Inc.). For multiple alignments, the default values cancorrespond to GAP PENALTY=10 and GAP LENGTH PENALTY=10. Defaultparameters for pairwise alignments and calculation of percent identityof protein sequences using the Clustal method can be KTUPLE=1, GAPPENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. For nucleic acids, theseparameters can be KTUPLE=2, GAP PENALTY=5, WINDOW=4 and DIAGONALSSAVED=4. Additionally, the Clustal W method of alignment can be used(described by Higgins and Sharp, CABIOS. 5:151-153 (1989); Higgins, D.G. et al., Comput. Appl. Biosci. 8:189-191(1992); Thompson, J. D. et al,Nucleic Acids Research, 22 (22): 4673-4680, 1994) and found in theMEGALIGN v8.0 program of the LASERGENE bioinformatics computing suite(DNASTAR Inc.). Default parameters for multiple alignment(protein/nucleic acid) can be: GAP PENALTY=10/15, GAP LENGTHPENALTY=0.2/6.66, Delay Divergent Seqs (%)=30/30, DNA TransitionWeight=0.5, Protein Weight Matrix=Gonnet Series, DNA Weight Matrix=IUB.

Various polypeptide amino acid sequences and polynucleotide sequencesare disclosed herein as features of certain embodiments. Variants ofthese sequences that are at least about 70-85%, 85-90%, or 90%-95%identical to the sequences disclosed herein can be used or referenced.Alternatively, a variant amino acid sequence or polynucleotide sequencecan have at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 99% identity with a sequence disclosed herein. Avariant amino acid sequence or polynucleotide sequence herein has thesame function/activity of the disclosed sequence, or at least about 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% of the function/activity of the disclosedsequence. Any polypeptide amino acid sequence disclosed herein notbeginning with a methionine can typically further comprise at least astart-methionine at the N-terminus of the amino acid sequence. Incontrast, any polypeptide amino acid sequence disclosed herein beginningwith a methionine can optionally lack such a methionine residue.

The terms “aligns with”, “corresponds with”, and the like can be usedinterchangeably herein. Some embodiments herein relate to aglucosyltransferase comprising at least one amino acid substitution at aposition corresponding with at least one particular amino acid residueof SEQ ID NO:62. An amino acid position of a glucosyltransferase orsubsequence thereof (e.g., catalytic domain or catalytic domain plusglucan-binding domains) (can refer to such an amino add position orsequence as a “query” position or sequence) can be characterized tocorrespond with a particular amino acid residue of SEQ ID NO:62 (canrefer to such an amino acid position or sequence as a “subject” positionor sequence) if (1) the query sequence can be aligned with the subjectsequence (e.g., where an alignment indicates that the query sequence andthe subject sequence [or a subsequence of the subject sequence] are atleast about 30%, 40%, 50%, 60%, 70%, 80%, or 90% identical), and (2) ifthe query amino acid position directly aligns with (directly lines upagainst) the subject amino acid position in the alignment of (1). Ingeneral, one can align a query amino acid sequence with a subjectsequence (SEQ ID NO:62 or a subsequence of SEQ ID NO:62) using anyalignment algorithm, tool and/or software described disclosed herein(e.g., BLASTP, ClustalW, ClustalV, Clustal-Omega, EMBOSS) to determinepercent identity. Just for further example, one can align a querysequence with a subject sequence herein using the Needleman-Wunschalgorithm (Needleman and Wunsch, J. Mol. Biol. 48:443-453, 1970) asimplemented in the Needle program of the European Molecular Biology OpenSoftware Suite (EMBOSS [e.g., version 5.0.0 or later], Rice et al.,Trends Genet. 16:276-277, 2000). The parameters of such an EMBOSSalignment can comprise, for example: gap open penalty of 10, gapextension penalty of 0.5, EBLOSUM62 (EMBOSS version of BLOSUM62)substitution matrix.

The numbering of particular amino acid residues of SEQ ID NO:62 herein(e.g., Leu-513, Pro-550, Ser-553, Asn-557, Asn-558, Trp-571, Asn-573,Asp-575, Lys-578, Asn-581, Thr-585, Gln-616) is with respect to thefull-length amino acid sequence of SEQ ID NO:62. The first amino acid(i.e., position 1, Met-1) of SEQ ID NO:62 is at the start of the signalpeptide. Unless otherwise disclosed, substitutions herein are withrespect to the full-length amino acid sequence of SEQ ID NO:62.

A “non-native glucosyltransferase” herein (“mutant”, “variant”,“modified” and like terms can likewise be used to describe such aglucosyltransferase) has at least one amino acid substitution at aposition corresponding with a particular amino acid residue of SEQ IDNO:62. Such at least one amino acid substitution typically is in placeof the amino acid residue(s) that normally (natively) occurs at the sameposition in the native counterpart (parent) of the non-nativeglucosyltransferase (i.e., although SEQ ID NO:62 is used as a referencefor position, an amino acid substitution herein is with respect to thenative counterpart of a non-native glucosyltransferase) (consideredanother way, when aligning the sequence of a non-nativeglucosyltransferase with SEQ ID NO:62, determining whether asubstitution exists at a particular position does not dependin-and-of-itself on the respective amino acid residue in SEQ ID NO:62,but rather depends on what amino acid exists at the subject positionwithin the native counterpart of the non-native glucosyltransferase).The amino acid normally occurring at the relevant site in the nativecounterpart glucosyltransferase often (but not always) is the same as(or conserved with) the particular amino acid residue of SEQ ID NO:62for which the alignment is made. A non-native glucosyltransferaseoptionally can have other amino acid changes (mutations, deletions,and/or insertions) relative to its native counterpart sequence.

It may be instructive to illustrate a substitution/alignment herein. SEQID NO:12 (GTF 0544) is a truncated form of a Streptococcus sobrinusglucosyltransferase. It is noted that Leu-193 of SEQ ID NO:12corresponds with Leu-373 of SEQ ID NO:62 (alignment not shown). If SEQID NO:12 is mutated at position 193 to substitute the Leu residue with adifferent residue (e.g., Gln), then it can be stated that the position193-mutated version of SEQ ID NO:12 represents a non-nativeglucosyltransferase having an amino acid substitution at a positioncorresponding with Leu-373 of SEQ ID NO:62, for example.

The term “isolated” means a substance (or process) in a form orenvironment that does not occur in nature. Non-limiting examples ofisolated substances include (1) any non-naturally occurring substance(e.g., a non-native glucosyltransferase herein), (2) any substanceincluding, but not limited to, any host cell, enzyme, variant, nucleicacid, protein, peptide, cofactor, or carbohydrate/saccharide that is atleast partially removed from one or more or all of the naturallyoccurring constituents with which it is associated in nature; (3) anysubstance modified by the hand of man relative to that substance foundin nature (e.g., a non-native glucosyltransferase herein); or (4) anysubstance modified by increasing the amount of the substance relative toother components with which it is naturally associated. It is believedthat the embodiments (e.g., enzymes and reaction compositions) disclosedherein are synthetic/man-made (could not have been made except for humanintervention/involvement), and/or have properties that are not naturallyoccurring.

The term “increased” as used herein can refer to a quantity or activitythat is at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%,12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 50%, 100%, or 200% morethan the quantity or activity for which the increased quantity oractivity is being compared. The terms “increased”, “elevated”,“enhanced”, “greater than”, “improved” and the like are usedinterchangeably herein. These terms can be used to characterize the“over-expression” or “up-regulation” of a polynucleotide encoding aprotein, for example.

While advances have been made in producing insoluble glucan polymersusing glucosyltransferase enzymes, less attention appears to have beendrawn to modulating the molecular weight of insoluble glucan productssynthesized by such enzymes. Addressing this technological gap,disclosed herein are glucosyltransferases with modified amino acidsequences that produce lower molecular weight insoluble glucan products.

Certain embodiments of the present disclosure concern a non-nativeglucosyltransferase comprising at least one amino acid substitution at aposition corresponding with amino acid residue(s) Leu-513, Pro-550,Ser-553, Asn-557, Asn-558, Trp-571, Asn-573, Asp-575, Lys-578, Asn-581,Thr-585, or Gln-616 of SEQ ID NO:62, wherein the non-nativeglucosyltransferase synthesizes insoluble alpha-glucan comprisingalpha-1,3-glycosidic linkages, and the molecular weight of the insolublealpha-glucan is lower than the molecular weight of insolublealpha-glucan synthesized by a second glucosyltransferase that onlydiffers from the non-native glucosyltransferase at the substitutionposition(s). Thus, in general, mutant glucosyltransferase enzymes aredisclosed herein that can synthesize lower molecular weight insolublealpha-glucan having alpha-1,3 glycosidic linkages.

A non-native glucosyltransferase herein synthesizes insolublealpha-glucan comprising alpha-1,3-glycosidic linkages. In some aspects,at least about 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%,41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%,55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 69%,70%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.5%, or 100% of the glycosidic linkages of such analpha-glucan can be alpha-1,3 linkages. The linkage profile of aninsoluble alpha-glucan can optionally be characterized as having a rangebetween any two of these values. The other linkages in any of theseaspects having 30%-99.5% alpha-1,3 linkages can be alpha-1,6, and/or notinclude any alpha-1,4 or alpha-1,2 linkages, for example.

Insoluble alpha-glucan in some aspects can have, for example, less than10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0.5% of alpha-1,2 oralpha-1,4 glycosidic linkages. In another embodiment, an insolublealpha-glucan only has alpha-1,3 and optionally alpha-1,6 linkages (i.e.,no alpha-1,2 or alpha-1,4 linkages). In aspects in which insolublealpha-glucan comprises 50% alpha-1,3 glycosidic linkages, such glucantypically does not comprise alternan (alternating alpha-1,3 and -1,6linkages).

Insoluble alpha-glucan in some aspects can be linear/unbranched (nobranch points). Alternatively, there can be branches in an insolublealpha-glucan herein. For example, an insoluble alpha-glucan can haveless than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0.5% branchpoints as a percent of the linkages in the polymer.

In certain aspects, an insoluble alpha-glucan can have a molecularweight in DPw or DPn of less than about 300. For example, the DPw or DPncan be about, or less than about, 300, 290, 280, 270, 260, 250, 240,230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100,95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 29, 28, 27, 26,25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, or 11. Themolecular weight of an insoluble alpha-glucan can optionally beexpressed as a range between any two of these values (e.g., 11-25,12-25, 11-22, 12-22, 11-20, 12-20, 20-300, 20-200, 20-150, 20-100,20-75, 30-300, 30-200, 30-150, 30-100, 30-75, 50-300, 50-200, 50-150,50-100, 50-75, 75-300, 75-200, 75-150, 75-100, 100-300, 100-200,100-150, 150-300, 150-200, 200-300). Molecular weight herein can bemeasured following any suitable method, including those methodsdisclosed in the present Examples (below) or as disclosed in U.S. Pat.Appl. Publ. Nos. 2017/0002335, 2015/0064748, or 2015/0232819, forexample.

Alpha-glucan herein is insoluble in non-caustic aqueous systems, such asthose conditions of a glucosyltransferase reaction herein (e.g., pH 4-8,see below). In general, the solubility of a glucan polymer in aqueoussettings herein is related to its linkage profile, molecular weight,and/or degree of branching. For example, alpha-1,3-glucan with 95% 1,3linkages is generally insoluble at a DPw of 8 and above in aqueousconditions at 20° C. In general, as molecular weight increases, thepercentage of alpha-1,3 linkages required for alpha-1,3-glucaninsolubility decreases.

Any of the foregoing linkage profiles and/or molecular weight profiles,for example, can be combined herein to appropriately characterize aninsoluble alpha-glucan product of a non-native glucosyltransferase ofthe present disclosure.

A non-native glucosyltransferase, for example, can comprise the aminoacid sequence of any glucosyltransferase disclosed in the followingpublications that is capable of producing insoluble alpha-glucan aspresently disclosed, but with the exception that the non-nativeglucosyltransferase comprises at least one amino acid substitution at aposition corresponding with amino acid residue Leu-513, Pro-550,Ser-553, Asn-557, Asn-558, Trp-571, Asn-573, Asp-575, Lys-578, Asn-581,Thr-585, or Gln-616 of SEQ ID NO:62: U.S. Pat. Nos. 7,000,000 and8,871,474; and U.S. Patent Appl. Publ. Nos. 2015/0232819 and2017/0002335, all of which are incorporated herein by reference. In someaspects, such a non-native glucosyltransferase (i) has at least one ofthe foregoing substitutions, and (ii) comprises an amino acid sequencethat is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 99.5% identical to the amino acid sequence of the respectivecounterpart/parent glucosyltransferase not having the at least onesubstitution.

In some aspects, a non-native glucosyltransferase (i) comprises at leastone amino acid substitution at a position corresponding with amino acidresidue Leu-513, Pro-550, Ser-553, Asn-557, Asn-558, Trp-571, Asn-573,Asp-575, Lys-578, Asn-581, Thr-585, or Gln-616 of SEQ ID NO:62, and (ii)comprises, or consists of, an amino acid sequence that is at least about90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical toSEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 26, 28, 30, 34, or 59.Certain information regarding insoluble alpha-glucan products ofglucosyltransferases with most of these amino acid sequences is providedin Table 2.

TABLE 2 GTF Enzymes and Related Alpha-Glucan Products^(a) Linkages SEQID Reducing Insoluble % alpha- % alpha- GTF ID NO. Sugars Product 1,31,6 DPn 0874 2 yes yes 100 0 60 6855 4 yes yes 100 0 440 2379 6 yes yes37 63 310 7527 8 yes yes 100 0 440 1724 10 yes yes 100 0 250 0544 12 yesyes 62 36 980 5926 14 yes yes 100 0 260 4297 16 yes yes 31 67 800 561818 yes yes 34 66 1020 2765 20 yes yes 100 0 280 0427 26 yes yes 100 0120 2919 28 yes yes 100 0 250 2678 30 yes yes 100 0 390 3929 34 yes yes100 0 280 ^(a)GTF reactions and product analyses were performed asfollows. Reactions were prepared comprising sucrose (50 g/L), potassiumphosphate buffer (pH 6.5, 20 mM) and a GTF enzyme (2.5% bacterial cellextract by volume; extracts prepared according to U.S. Appl. Publ. No.2017/0002335, in a manner similar to procedure disclosed in U.S. Pat.No. 8,871,474). After 24-30 hours at 22-25° C., insoluble product washarvested by centrifugation, washed three times with water, washed oncewith ethanol, and dried at 50° C. for 24-30 hours. Approximate linkagesand DPn are shown for each insoluble product. Linkages and DPn weredetermined by ¹³C NMR and SEC, respectively.

In some aspects, a non-native glucosyltransferase (i) comprises at leastone amino acid substitution at a position corresponding with amino acidresidue Leu-513, Pro-550, Ser-553, Asn-557, Asn-558, Trp-571, Asn-573,Asp-575, Lys-578, Asn-581, Thr-585, or Gln-616 of SEQ ID NO:62, and (ii)comprises, or consists, of a glucosyltransferase catalytic domain thatis at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or99.5% identical to SEQ ID NO:66 (amino acid residues 55-960 of SEQ IDNO:4), residues 54-957 of SEQ ID NO:65, residues 55-960 of SEQ ID NO:30,residues 55-960 of SEQ ID NO:28, or residues 55-960 of SEQ ID NO:20.Such a non-native glucosyltransferase, for instance, is believed to beable to produce alpha-glucan that is water-insoluble and comprise atleast about 50% (e.g., ≥90% or ≥95%) alpha-1,3 linkages. It is notedthat a glucosyltransferase with amino acid positions 54-957 of SEQ IDNO:65 can produce alpha-1,3-glucan with 100% alpha-1,3 linkages (datanot shown, refer to Table 6 of U.S. Pat. Appl. Publ. No. 2017/0002335,which is incorporated herein by reference), for example. It is furthernoted that SEQ ID NOs:65 (GTF 7527), 30 (GTF 2678), 4 (GTF 6855), 28(GTF 2919), and 20 (GTF 2765) each represent a glucosyltransferase that,compared to its respective wild type counterpart, lacks the signalpeptide domain and all or a substantial portion of the variable domain.Thus, each of these glucosyltransferase enzymes has a catalytic domainfollowed by a glucan-binding domain. The approximate location ofcatalytic domain sequences in these enzymes is as follows: 7527(residues 54-957 of SEQ ID NO:65), 2678 (residues 55-960 of SEQ IDNO:30), 6855 (SEQ ID NO:66, which is residues 55-960 of SEQ ID NO:4),2919 (residues 55-960 of SEQ ID NO:28), 2765 (residues 55-960 of SEQ IDNO:20). The amino acid sequences of the catalytic domains (approx.) ofGTFs 2678, 6855, 2919 and 2765 have about 94.9%, 99.0%, 95.5% and 96.4%identity, respectively, with the approximate catalytic domain sequenceof GTF 7527 (i.e., amino acids 54-957 of SEQ ID NO:65). Each of theseparticular glucosyltransferases (GTFs 2678, 6855, 2919 and 2765) canproduce alpha-1,3-glucan with 100% alpha-1,3 linkages and a DPw of atleast 400 (data not shown, refer to Table 4 of U.S. Pat. Appl. Publ. No.2017/0002335). Based on this activity, and the relatedness (high percentidentity) of the foregoing catalytic domains, it is contemplated that anon-native glucosyltransferase herein having one of the foregoingcatalytic domains further with at least one amino acid substitution aspresently disclosed can produce lower molecular weight insolublealpha-glucan comprising at least about 50% (e.g., ≥90% or ≥95%)alpha-1,3 linkages.

In some aspects, a non-native glucosyltransferase (i) comprises at leastone amino acid substitution at a position corresponding with amino acidresidue Leu-513, Pro-550, Ser-553, Asn-557, Asn-558, Trp-571, Asn-573,Asp-575, Lys-578, Asn-581, Thr-585, or Gln-616 of SEQ ID NO:62, and (ii)comprises or consists of an amino acid sequence that is at least about40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%,54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%,69%, 70%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO:62 or a subsequencethereof such as SEQ ID NO:4 (without start methionine thereof) or SEQ IDNO:66 (positions 55-960 of SEQ ID NO:4, approximate catalytic domain).

Although it is believed that a non-native glucosyltransferase in certainaspects need only have a catalytic domain, the non-nativeglucosyltransferase can be comprised within a larger amino acidsequence. For example, a catalytic domain may be linked at itsC-terminus to a glucan-binding domain, and/or linked at its N-terminusto a variable domain and/or signal peptide.

Although amino acid substitutions in a non-native glucosyltransferaseare generally disclosed herein with respect to the correspondingpositions in SEQ ID NO:62, such substitutions can alternatively bestated simply with respect to its position number in the sequence of thenon-native glucosyltransferase itself, as convenience may dictate.

Still further examples of non-native glucosyltransferases can be any asdisclosed herein and that include 1-300 (or any integer there between[e.g., 10, 15, 20, 25, 30, 35, 40, 45, or 50]) residues on theN-terminus and/or C-terminus. Such additional residues may be from acorresponding wild type sequence from which the glucosyltransferaseenzyme is derived, or may be a heterologous sequence such as an epitopetag (at either N- or C-terminus) or a heterologous signal peptide (atN-terminus), for example. A non-native glucosyltransferase hereintypically lacks an N-terminal signal peptide; such an enzyme canoptionally be characterized as being mature if its signal peptide wasremoved during a secretion process.

A non-native glucosyltransferase herein can be derived from anymicrobial source, for example, such as bacteria. Examples of bacterialglucosyltransferases are those derived from a Streptococcus species,Leuconostoc species, or Lactobacillus species. Examples of Streptococcusspecies include S. salivarius, S. sobrinus, S. dentirousetti, S. downei,S. mutans, S. oralis, S. gallolyticus and S. sanguinis. Examples ofLeuconostoc species include L. mesenteroides, L. amelibiosum, L.argentinum, L. carnosum, L. citreum, L. cremoris, L. dextranicum and L.fructosum. Examples of Lactobacillus species include L. acidophilus, L.delbrueckii, L. helveticus, L. salivarius, L. casei, L. curvatus, L.plantarum, L. sakei, L. brevis, L. buchneri, L. fermentum and L.reuteri.

A non-native glucosyltransferase herein can be prepared by fermentationof an appropriately engineered microbial strain, for example.Recombinant enzyme production by fermentation is well known in the artusing microbial species such as E. coli, Bacillus strains (e.g., B.subtilis), Ralstonia eutropha, Pseudomonas fluorescens, Saccharomycescerevisiae, Pichia pastoris, Hansenula polymorpha, and species ofAspergillus (e.g., A. awamon) and Trichoderma (e.g., T. reesei) (e.g.,see Adrio and Demain, Biomolecules 4:117-139, 2014, which isincorporated herein by reference). A nucleotide sequence encoding anon-native glucosyltransferase amino acid sequence is typically linkedto a heterologous promoter sequence to create an expression cassette forthe enzyme, and/or is codon-optimized accordingly. Such an expressioncassette may be incorporated in a suitable plasmid or integrated intothe microbial host chromosome, using methods well known in the art. Theexpression cassette may include a transcriptional terminator nucleotidesequence following the amino acid coding sequence. The expressioncassette may also include, between the promoter sequence andglucosyltransferase amino acid coding sequence, a nucleotide sequenceencoding a signal peptide (e.g., heterologous signal peptide) that isdesigned for direct secretion of the glucosyltransferase enzyme. At theend of fermentation, cells may be ruptured accordingly (generally when asignal peptide for secretion is not employed) and theglucosyltransferase enzyme can be isolated using methods such asprecipitation, filtration, and/or concentration. Alternatively, a lysateor extract comprising a glucosyltransferase can be used without furtherisolation. If the glucosyltransferase was secreted (i.e., it is presentin the fermentation broth), it can optionally be used as isolated from,or as comprised in, the fermentation broth. The activity of aglucosyltransferase enzyme can be confirmed by biochemical assay, suchas measuring its conversion of sucrose to glucan polymer.

A non-native glucosyltransferase herein can comprise at least one aminoacid substitution at a position corresponding with amino acid residueLeu-513, Pro-550, Ser-553, Asn-557, Asn-558, Trp-571, Asn-573, Asp-575,Lys-578, Asn-581, Thr-585, or Gln-616 of SEQ ID NO:62. In some aspects,a non-native glucosyltransferase comprises at least one amino acidsubstitution at a position corresponding with amino acid residueLeu-513, Ser-553, Asn-573, Asp-575, Lys-578, or Gln-616 of SEQ ID NO:62.In some aspects, the amino acid substitution at a position correspondingwith amino acid residue Leu-513 of SEQ ID NO:62 can be with an Ala, Cys,Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr,Val, Trp, or Tyr residue. In some aspects, the amino acid substitutionat a position corresponding with amino acid residue Pro-550 of SEQ IDNO:62 can be with a Leu, Val, or Ile residue (e.g., Leu or Val). In someaspects, the amino acid substitution at a position corresponding withamino acid residue Ser-553 of SEQ ID NO:62 can be with an Ala, Cys, Glu,Phe, His, Ile, Met, Asn, Arg, Thr, Val, or Tyr residue. In some aspects,the amino acid substitution at a position corresponding with amino acidresidue Asn-557 of SEQ ID NO:62 can be with a Glu, Gln, Ile, Thr, Asp,Asn, Leu, Val, or Ser residue (e.g., Glu, Gln, Ile, or Thr). In someaspects, the amino acid substitution at a position corresponding withamino acid residue Asn-558 of SEQ ID NO:62 can be with an Asp or Gluresidue (e.g., Asp). In some aspects, the amino acid substitution at aposition corresponding with amino acid residue Trp-571 of SEQ ID NO:62can be with a Val, Asp, Cys, Ile, Leu, Glu, or Ser residue (e.g., Val,Asp, or Cys). In some aspects, the amino acid substitution at a positioncorresponding with amino acid residue Asn-573 of SEQ ID NO:62 can bewith an Ala, Asp, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Thr, Val, orTrp residue. In some aspects, the amino acid substitution at a positioncorresponding with amino acid residue Asp-575 of SEQ ID NO:62 can bewith a Ala, Cys, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg,Ser, Val, Trp, or Tyr residue. In some aspects, the amino acidsubstitution at a position corresponding with amino acid residue Lys-578of SEQ ID NO:62 can be with an Ala, Cys, Asp, Glu, Phe, Gly, His, Ile,Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr residue. Insome aspects, the amino acid substitution at a position correspondingwith amino acid residue Asn-581 of SEQ ID NO:62 can be with a Pro or Glyresidue (e.g., Pro). In some aspects, the amino acid substitution at aposition corresponding with amino acid residue Thr-585 of SEQ ID NO:62can be with Pro or Gly residue (e.g., Pro). In some aspects, the aminoacid substitution at a position corresponding with amino acid residueGln-616 of SEQ ID NO:62 can be with an Ala, Cys, Asp, Glu, Gly, His,Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr residue.

Suitable substitution sites, and examples of particular substitutions atthese sites, can include those as listed in Table 3 in Example 1 (below)that are associated with a decrease in the molecular weight (DPw) ofinsoluble alpha-1,3-glucan product by about, or at least about, 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or85%, for example. The foregoing substitutions as listed in Table 3 areas they correspond with the listed residue position number in SEQ IDNO:62.

A non-native glucosyltransferase herein can comprise one, two, three,four, five, six, seven, or more of the presently disclosed amino acidsubstitutions, for instance. For example, a non-nativeglucosyltransferase with two or more of the presently disclosed aminoacid substitutions can comprise at least one amino acid substitution ata position corresponding with (i) amino acid residue Pro-550, Asn-557,Asn-558, Asn-581, or Thr-585 of SEQ ID NO:62, and/or (ii) amino acidresidue Leu-513, Ser-553, Asn-573, Asp-575, Lys-578, or Gln-616 of SEQID NO:62. Examples of substitution positions are at P550(L) and N557(I),P550(L) and N581(P), N557(I) and N581(P), 5553(C) and N558(D), 5553(C)and D575(V), S553(C) and T585(P), N558(D) and T585(P), D575(V) andT585(P), N558(D) and D575(V), P550(V) and S553(R), P550(V) and N581(P),P550(V) and T585(P), S553(R) and N581(P), S553(R) and T585(P), N581(P)and T585(P), P550(L) and S553(F), P550(L) and N581(P), S553(F) andN581(P), P550(V) and N557(E), P550(V) and T585(P) N557(E) and T585(P),where substituting amino acid residues are listed parenthetically asexamples; one or more substitutions in addition to any at the foregoingposition pairs can optionally be at position P550(L, V), N557(I, E),N581(P), 5553 (any herein such as C, R, F), N558(D), D575 (any hereinsuch as V, A), T585(P), L513 (any herein), N573 (any herein such as I,V, P), K578 (any herein such as N, D, R), Q616 (any herein), W571(V, D,C), and/or any other position herein accordingly, where substitutingamino acid residues are listed parenthetically as examples.

Simply for illustration purposes, a non-native glucosyltransferaseherein can comprise a combination of amino acid substitutions atpositions as shown in Table A (i-lxiii), where each substitutionposition corresponds with the respective amino acid position number inSEQ ID NO:62. The substituting amino acid residues in Table A are listedparenthetically as examples. In some aspects, a non-nativeglucosyltransferase can comprise a combination of amino acidsubstitutions as shown in Table A, where the substituting amino acidsare those shown parenthetically in Table A.

TABLE A Examples of Amino Acid Substitution CombinationsSubstitution^(a) Combinations i P550(L) N557(I) N581(P) ii L535(P)S553(C) N558(D) D575(V) T585(P) K697(R) iii P550(V) S553(R) N581(P)T585(P) iv P550(L) S553(F) N581(P) v P550(V) N557(E) T585(P) vi P550(L)N557(E) D575(V) T585(P) vii L538(P) P550(L) S553(Y) viii P544(L) P550(V)S553(C) N573(I) T585(P) S589(G) ix P550(V) G576(D) T585(P) x P550(L)N558(D) T585(P) T679(I) xi P550(L) N557(E) T585(P) S589(G) xii P550(L)N557(E) T569(L) N581(P) xiii P550(L) N557(E) T569(L) T585(P) xiv P550(V)S553(T) N558(D) T585(P) G730(D) xv E577(G) P550(L) N557(I) T569(L)N573(I) xvi P550(L) S553(C) D575(A) T585(P) S589(G) xvii S553(R) N573(V)K578(N) S631(G) T660(A) xviii P550(V) S553(R) W571(V) G576(D) xixP550(V) N557(E) K578(D) T585(P) xx P550(V) N558(D) N573(P) T585(P) xxiP550(L) N558(D) W571(V) N581(P) K593(E) xxii P550(V) S553(E) N581(P)xxiii P550(L) N573(I) T585(P) W725(R) xxiv P550(L) N557(I) N573(P) xxvN557(E) N573(V) N581(P) xxvi P550(L) N557(I) G576(D) Q643(L) xxviiP550(V) S553(N) T585(P) V586(G) S710(G) xxviii P550(V) S553(C) D575(A)T585(P) xxix S553(R) N573(V) K578(N) S631(G) T660(A) xxx P550(L) S553(K)D575(A) Y580(H) xxxi P550(V) D575(A) T585(P) S589(G) K713(E) xxxiiP550(V) S553(N) N573(I) Y693(C) xxxiii P544(L) P550(L) N557(E) N573(I)T585(P) xxxiv P550(L) N558(D) W571(V) N581(P) T585(P) xxxv S504(G)P550(V) N557(Q) N581(P) xxxvi P550(L) S553(R) D575(A) xxxvii P550(V)N558(D) W571(D) D575(A) T585(P) xxxviii P544(L) P550(V) N557(Q) N581(P)xxxix P550(V) S553(K) T585(P) xl P550(V) S553(N) T585(P) xli P550(L)T569(L) N573(I) xlii P550(L) N558(D) D575(V) xliii L537(P) P550(L)N558(D) N573(I) xliv P550(L) S553(C) W571(C) G576(D) T585(P) xlv P550(L)N557(Q) W571(C) G576(D) T585(P) xlvi P550(L) N558(D) W571(V) N581(P)xlvii A542(V) P550(V) N558(D) W571(V) T585(P) xlviii P550(V) N558(D)W571(D) G576(D) xlix P550(V) S553(N) N573(I) l P550(V) N557(Q) D575(V)A669(T) li P550(V) N581(P) I636(T) lii P550(V) N557(E) N581(P) liiiP550(V) N573(I) T585(P) liv P550(L) S553(K) N558(D) K578(R) Y700(N) lvP550(V) S553(T) N558(D) W571(V) lvi P514(L) P550(V) N557(Q) T585(P)D602(N) lvii P550(L) N557(I) T569(A) G576(D) lviii P550(V) N557(T)N558(D) W571(D) lix P550(L) N557(E) D575(A) T585(P) lx I545(V) P550(V)N557(Q) T585(P) D638(N) lxi P544(L) P550(V) N557(I) T585(P) lxii Y518(C)P550(V) N581(P) T585(P) lxiii P550(L) N557(E) D575(A) ^(a)Amino acidresidues listed parenthetically are examples of substituting amino acidresidues.

A non-native glucosyltransferase with one or more amino acidsubstitutions herein can be based on any of a variety ofglucosyltransferase amino acid sequences as presently disclosed, forexample. Simply for illustration purposes, examples of such a non-nativeglucosyltransferase include those with a single amino acid substitution(e.g., at a position corresponding with amino acid residue Leu-513,Pro-550, Ser-553, Asn-557, Asn-558, Trp-571, Asn-573, Asp-575, Lys-578,Asn-581, Thr-585, or Gln-616 of SEQ ID NO:62) or a combination of aminoacid substitutions (e.g., any of embodiments i-lxiii of Table A) andthat comprise or consist of an amino acid sequence that is at leastabout 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5%identical to SEQ ID NO:65 (optionally without the start methionine ofSEQ ID NO:65) or residues 54-957 of SEQ ID NO:65, SEQ ID NO:30(optionally without the start methionine of SEQ ID NO:30) or residues55-960 of SEQ ID NO:30, SEQ ID NO:4 (optionally without the startmethionine of SEQ ID NO:4) or SEQ ID NO:66 (residues 55-960 of SEQ IDNO:4), SEQ ID NO:28 (optionally without the start methionine of SEQ IDNO:28) or residues 55-960 of SEQ ID NO:28, or SEQ ID NO:20 (optionallywithout the start methionine of SEQ ID NO:20) or residues 55-960 of SEQID NO:20.

In some aspects, one or more substitutions are conserved ornon-conserved substitutions; such conservation (or not) can be, forinstance, with respect to the amino acid that occurs in the nativeglucosyltransferase from which the non-native glucosyltransferase isderived.

A non-native glucosyltransferase herein can synthesize insolublealpha-glucan comprising alpha-1,3-glycosidic linkages with a molecularweight lower than the molecular weight of insoluble alpha-glucancomprising alpha-1,3-glycosidic linkages synthesized by a secondglucosyltransferase (or, simply, “another” glucosyltransferase) (e.g.,parent glucosyltransferase) that only differs from the non-nativeglucosyltransferase at the substitution position(s). A secondglucosyltransferase herein, for example, can be comprised of all of, ormostly, native amino acid sequence. Thus, while a secondglucosyltransferase herein can be a native glucosyltransferase in someaspects, it can be a prior-modified glucosyltransferase in other aspects(e.g., a glucosyltransferase with one or more other amino acidsubstitutions differing from the substitution[s] of the presentdisclosure). In some embodiments, a second glucosyltransferase to whicha non-native glucosyltransferase is compared has a native amino acidresidue(s) at the substitution position(s). Determining whether an aminoacid residue is native can be done by comparing the secondglucosyltransferase amino acid sequence to the native/wild typeglucosyltransferase amino acid sequence from which the secondglucosyltransferase is derived.

In some aspects, a non-native glucosyltransferase herein can synthesizeinsoluble alpha-glucan with a molecular weight that is about, or atleast about, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, or 90% lower than the molecular weight ofinsoluble alpha-glucan synthesized by a second glucosyltransferase. Sucha determination can be made with respect to any glucan synthesisreaction/process as disclosed herein (e.g., taking into account initialsucrose conc., temperature, pH, and/or reaction time), and using anysuitable measurement technique (e.g., SEC). Typically, a comparisonbetween non-native and second glucosyltransferases herein can be madeunder identical or similar reaction conditions. The molecular weight ofinsoluble alpha-glucan can be expressed as DPw, for example. Molecularweight can optionally be expressed as DP in embodiments regardinginsoluble glucan with 20 or less monomeric units, for example.

Some embodiments disclosed herein concern a polynucleotide comprising anucleotide sequence that encodes a non-native glucosyltransferase aspresently disclosed (e.g., a non-native glucosyltransferase comprisingat least one amino acid substitution at a position corresponding withamino acid residue Leu-513, Pro-550, Ser-553, Asn-557, Asn-558, Trp-571,Asn-573, Asp-575, Lys-578, Asn-581, Thr-585, or Gln-616 of SEQ IDNO:62). Optionally, one or more regulatory sequences are operably linkedto the nucleotide sequence, and preferably a promoter sequence isincluded as a regulatory sequence.

A polynucleotide comprising a nucleotide sequence encoding a non-nativeglucosyltransferase herein can be a vector or construct useful fortransferring a nucleotide sequence into a cell, for example. Examples ofa suitable vector/construct can be selected from a plasmid, yeastartificial chromosome (YAC), cosmid, phagemid, bacterial artificialchromosome (BAC), virus, or linear DNA (e.g., linear PCR product). Apolynucleotide sequence in some aspects can be capable of existingtransiently (i.e., not integrated into the genome) or stably (i.e.,integrated into the genome) in a cell. A polynucleotide sequence in someaspects can comprise, or lack, one or more suitable marker sequences(e.g., selection or phenotype marker).

A polynucleotide sequence in certain embodiments can comprise one ormore regulatory sequences operably linked to the nucleotide sequenceencoding a non-native glucosyltransferase. For example, a nucleotidesequence encoding a non-native glucosyltransferase may be in operablelinkage with a promoter sequence (e.g., a heterologous promoter). Apromoter sequence can be suitable for expression in a cell (e.g.,bacterial cell such as E. coli or Bacillus; eukaryotic cell such as afungus, yeast, insect, or mammalian cell) or in an in vitro proteinexpression system, for example. Examples of other suitable regulatorysequences include transcription terminator sequences.

Some aspects herein are drawn to a cell comprising a polynucleotidesequence as presently disclosed; such a cell can be any type disclosedherein (e.g., bacterial cell such as E. coli or Bacillus; eukaryoticcell such as a fungus, yeast, insect, or mammalian cell). A cell canoptionally express a non-native glucosyltransferase encoded by thepolynucleotide sequence. In some aspects, the polynucleotide sequenceexists transiently (i.e., not integrated into the genome) or stably(i.e., integrated into the genome) in the cell.

Some embodiments disclosed herein concern a reaction compositioncomprising water, sucrose, and one or more non-nativeglucosyltransferases herein (e.g., a non-native glucosyltransferasecomprising at least one amino acid substitution at a positioncorresponding with amino acid residue Leu-513, Pro-550, Ser-553,Asn-557, Asn-558, Trp-571, Asn-573, Asp-575, Lys-578, Asn-581, Thr-585,or Gln-616 of SEQ ID NO:62). Such a reaction composition produces, atleast, insoluble alpha-glucan comprising alpha-1,3-glycosidic linkagesas disclosed.

The temperature of a reaction composition herein can be controlled, ifdesired, and can be about 5-50° C., 20-40° C., 30-40° C., 20-30° C.,20-25° C., 20° C., 25° C., 30° C., 35° C., or 40° C., for example.

The initial concentration of sucrose in a reaction composition hereincan be about 20-400 g/L, 75-175 g/L, or 50-150 g/L, for example. In someaspects, the initial sucrose concentration is about, or at least about,50, 75, 100, 150 or 200 g/L, or is about 50-600 g/L, 100-500 g/L, 50-100g/L, 100-200 g/L, 150-450 g/L, 200-450 g/L, or 250-600 g/L. “Initialconcentration of sucrose” refers to the sucrose concentration in areaction composition just after all the reaction components have beenadded/combined (e.g., at least water, sucrose, non-nativeglucosyltransferase enzyme).

The pH of a reaction composition in certain embodiments can be about4.0-9.0, 4.0-8.5, 4.0-8.0, 5.0-8.0, 5.5-7.5, or 5.5-6.5. In someaspects, the pH can be about 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, or8.0. The pH can be adjusted or controlled by the addition orincorporation of a suitable buffer, including but not limited to:phosphate, tris, citrate, or a combination thereof. The bufferconcentration in a reaction composition herein can be about 0.1-300 mM,0.1-100 mM, 10-100 mM, 10 mM, 20 mM, or 50 mM, for example.

A reaction composition can be contained within any vessel (e.g., aninert vessel/container) suitable for applying one or more of thereaction conditions disclosed herein. An inert vessel in some aspectscan be of stainless steel, plastic, or glass (or comprise two or more ofthese components) and be of a size suitable to contain a particularreaction. For example, the volume/capacity of an inert vessel (and/orthe volume of a reaction composition herein), can be about, or at leastabout, 1, 10, 50, 100, 500, 1000, 2500, 5000, 10000, 12500, 15000, or20000 liters. An inert vessel can optionally be equipped with a stirringdevice. Any of the foregoing features, for example, can be used tocharacterize an isolated reaction herein.

A reaction composition herein can contain one, two, or more differentglucosyltransferase enzymes, for example, just as long that at least oneof the enzymes is a non-native glucosyltransferase as presentlydisclosed. In some embodiments, only one or two glucosyltransferaseenzymes is/are comprised in a reaction composition. Aglucosyltransferase reaction herein can be, and typically is, cell-free(e.g., no whole cells present).

Any of the features disclosed herein (e.g., above and in the belowExamples) regarding a reaction composition can characterize appropriateaspects of a glucan production method herein, and vice versa.

The present disclosure also concerns a method for producing insolublealpha-glucan comprising alpha-1,3-glycosidic linkages, the methodcomprising: (a) contacting at least water, sucrose, and at least onenon-native glucosyltransferase as disclosed herein that producesinsoluble alpha-glucan comprising alpha-1,3-glycosidic linkages, wherebyinsoluble alpha-glucan comprising alpha-1,3-glycosidic linkages isproduced; and b) optionally, isolating the insoluble alpha-glucanproduced in step (a). Conducting such a method, which can optionally becharacterized as a glucan synthesis method, is typically also performedwhen conducting a reaction composition herein.

A glucan synthesis method as presently disclosed comprises contacting atleast water, sucrose, and a non-native glucosyltransferase herein thatproduces insoluble alpha-glucan comprising alpha-1,3-glycosidiclinkages. These and optionally other reagents can be added altogether orin any order as discussed below. This step can optionally becharacterized as providing a reaction composition comprising water,sucrose and a non-native glucosyltransferase enzyme that synthesizesinsoluble alpha-glucan comprising alpha-1,3-linkages. The contactingstep herein can be performed in any number of ways. For example, thedesired amount of sucrose can first be dissolved in water (optionally,other components may also be added at this stage of preparation, such asbuffer components), followed by addition of glucosyltransferase enzyme.The solution may be kept still, or agitated via stirring or orbitalshaking, for example. A glucan synthesis method can be performed bybatch, fed-batch, continuous mode, or by any variation of these modes.

Completion of a reaction in certain embodiments can be determinedvisually (e.g., no more accumulation of insoluble glucan), and/or bymeasuring the amount of sucrose left in the solution (residual sucrose),where a percent sucrose consumption of at least about 90%, 95%, or 99%can indicate reaction completion. A reaction of the disclosed processcan be conducted for about 1 hour to about, or at least about, 2, 4, 6,8, 10, 12, 14, 16, 18, 20, 22, 24, 36, 48, 60, 72, 96, 120, 144, or 168hours, for example.

The molecular weight of insoluble alpha-glucan produced in some aspectsof a glucan synthesis method herein can be about, or at least about, 5%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, or 90% lower than the molecular weight of insolublealpha-glucan synthesized by a second glucosyltransferase. Such molecularweight down-modulation in some aspects is achieved in a reactionconducted for about 36-60 hours (e.g., ˜48 hours).

Insoluble alpha-glucan comprising alpha-1,3-linkages produced in amethod herein can optionally be isolated. In certain embodiments,isolating insoluble alpha-glucan can include at least conducting a stepof centrifugation and/or filtration. Isolation can optionally furthercomprise washing insoluble alpha-glucan one, two, or more times withwater or other aqueous liquid, and/or drying the insoluble alpha-glucanproduct.

Any of the disclosed conditions for synthesizing insoluble alpha-glucan,such as the foregoing or those described in the below Examples, can beapplied to practicing a reaction composition as presently disclosed (andvice versa), and/or used to characterize features/activity of anon-native glucosyltransferase, accordingly.

In some aspects, an insoluble alpha-glucan product that has beenisolated (optionally characterized as “purified”) can be present in acomposition at a wt % (dry weight basis) of at least about 50%, 60%,70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%,99.5%, 99.8%, or 99.9%. Such isolated polymer can be used as aningredient/component in a product/application, for example.

The present disclosure also concerns a method of preparing apolynucleotide sequence encoding a non-native glucosyltransferaseherein. This method comprises:

(a) identifying a polynucleotide sequence encoding a parentglucosyltransferase that (i) comprises an amino acid sequence that is atleast about 40% identical to SEQ ID NO:4 or SEQ ID NO:66 (positions55-960 of SEQ ID NO:4), and (ii) synthesizes insoluble alpha-glucancomprising alpha-1,3-glycosidic linkages; and

(b) modifying the polynucleotide sequence identified in step (a) tosubstitute at least one amino acid of the parent glucosyltransferase ata position corresponding with amino acid residue Leu-513, Pro-550,Ser-553, Asn-557, Asn-558, Trp-571, Asn-573, Asp-575, Lys-578, Asn-581,Thr-585, or Gln-616 of SEQ ID NO:62, thereby providing a polynucleotidesequence encoding a non-native glucosyltransferase that synthesizesinsoluble alpha-glucan comprising alpha-1,3-glycosidic linkages andhaving a molecular weight that is lower than the molecular weight ofinsoluble alpha-glucan synthesized by the parent glucosyltransferase.

Such a method can optionally further comprise using a polynucleotideprepared in this manner in a method of expressing the non-nativeglucosyltransferase encoded by the polynucleotide. Such an expressionmethod can follow any heterologous protein expression method as known inthe art, for example. The present method of preparing a polynucleotidecan optionally alternatively be characterized as a method of decreasingthe product molecular weight of a glucosyltransferase.

A parent glucosyltransferase enzyme herein can comprise an amino acidsequence that is at least about 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%,48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%,62%, 63%, 64%, 65%, 66%, 67%, 69%, 70%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical toSEQ ID NO:4 (optionally without start methionine thereof) or SEQ IDNO:66 (positions 55-960 of SEQ ID NO:4, approximate catalytic domain),for example. It is noted simply for reference purposes that SEQ ID NO:4without its start methionine is a subsequence of SEQ ID NO:62.

Identification step (a) herein can, in some instances, compriseidentifying an amino acid sequence of a parent glucosyltransferaseenzyme. A polynucleotide sequence can be determined from this amino acidsequence according to the genetic code (codons), such as the geneticcode used in the species from which the parent glucosyltransferase wasidentified.

Identifying a polynucleotide encoding a parent glucosyltransferaseherein can be performed (a) in silico, (b) with a method comprising anucleic acid hybridization step, (c) with a method comprising a proteinsequencing step, and/or (d) with a method comprising a protein bindingstep, for example.

Regarding in silico detection, the amino acid sequences of candidateparent glucosyltransferase enzymes (and/or nucleotide sequences encodingsuch glucosyltransferase enzymes) stored in a computer or database(e.g., public databases such as GENBANK, EMBL, REFSEQ, GENEPEPT,SWISS-PROT, PIR, PDB) can be reviewed in silico to identify aglucosyltransferase enzyme comprising an amino acid sequence with apercent sequence identity as described above for a parentglucosyltransferase. Such review could comprise using any means known inthe art such as through use of an alignment algorithm or software asdescribed above (e.g., BLASTN, BLASTP, ClustalW, ClustalV,Clustal-Omega, EMBOSS).

Identifying a parent glucosyltransferase as disclosed above canoptionally be performed via a method comprising a nucleic acidhybridization step. Such a method can comprise using DNA hybridization(e.g., Southern blot, dot blot), RNA hybridization (e.g., northernblot), or any other method that has a nucleic acid hybridization step(e.g., DNA sequencing, PCR, RT-PCR, all of which may comprisehybridization of an oligonucleotide), for example. A polynucleotidesequence encoding SEQ ID NO:4 or a subsequence thereof (e.g., SEQ IDNO:66, which is positions 55-960 of SEQ ID NO:4) can be used as a probe,for example, in such a hybridization. Conditions and parameters forcarrying out hybridization methods in general are well known anddisclosed, for example, in Sambrook J, Fritsch E F and Maniatis T,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory:Cold Spring Harbor, N.Y. (1989); Silhavy T J, Bennan M L and Enquist LW, Experiments with Gene Fusions, Cold Spring Harbor Laboratory: ColdSpring Harbor, N.Y. (1984); Ausubel F M et al., Current Protocols inMolecular Biology, published by Greene Publishing Assoc. andWiley-Interscience, Hoboken, N.J. (1987); and Innis M A, Gelfand D H,Sninsky J J and White T J (Editors), PCR Protocols: A Guide to Methodsand Applications, Academic Press, Inc., San Diego, Calif. (1990).

Identifying a parent glucosyltransferase as disclosed above canoptionally be performed via a method comprising a protein sequencingstep. Such a protein sequencing step can comprise one or more proceduressuch as N-terminal amino acid analysis, C-terminal amino acid analysis,Edman degradation, or mass spectrometry, for example.

Identifying a parent glucosyltransferase as disclosed above canoptionally be performed via a method comprising a protein binding step.Such a protein binding step can be performed using an antibody thatbinds to a motif or epitope within SEQ ID NO:4 (e.g., within SEQ IDNO:66, which is positions 55-960 of SEQ ID NO:4), for example.

A polynucleotide identified in step (a) (i.e., before its modificationin step [b]) can, in some aspects, encode a glucosyltransferasecomprising an amino acid sequence that is identical to, or at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to, theamino acid sequence of any glucosyltransferase disclosed in Table 1. Analpha-glucan as produced by such a glucosyltransferase can be asdisclosed herein, for example.

A method of preparing a polynucleotide sequence encoding a non-nativeglucosyltransferase herein comprises step (b) of modifying thepolynucleotide sequence (encoding a parent glucosyltransferase)identified in step (a). Such modification substitutes at least one aminoacid of the parent glucosyltransferase at a position corresponding withamino acid residue Leu-513, Pro-550, Ser-553, Asn-557, Asn-558, Trp-571,Asn-573, Asp-575, Lys-578, Asn-581, Thr-585, or Gln-616 of SEQ ID NO:62.The non-native glucosyltransferase (encoded by the modifiedpolynucleotide sequence) resulting from such one or more substitutionscan be optionally be characterized as a “child glucosyltransferase”herein.

A suitable modification of a polynucleotide in step (b) can be madefollowing any DNA manipulation technique known in the art. Modifyingstep (b) can optionally be performed in silico, followed by synthesis ofthe polynucleotide sequence encoding a non-native glucosyltransferase.For example, a polynucleotide sequence identified in step (a) can bemanipulated in silico using a suitable sequence manipulationprogram/software (e.g., VECTOR NTI, Life Technologies, Carlsbad, Calif.;DNAStrider; DNASTAR, Madison, Wis.). Following such virtualmanipulation, the modified polynucleotide sequence can be artificiallysynthesized by any suitable technique (e.g., annealing-based connectionof oligonucleotides, or any technique disclosed in Hughes et al.,Methods Enzymol. 498:277-309, which is incorporated herein byreference). It should be appreciated that the foregoing methodology isnot believed to necessarily rely on having a pre-existing polynucleotide(encoding a parent glucosyltransferase) in hand.

Modifying step (b) can optionally be performed using a physical copy ofa polynucleotide sequence identified in step (a) encoding a parentglucosyltransferase. As an example, such a polynucleotide can serve as atemplate for amplification using primers designed in a manner such thatthe amplified product encodes a non-native glucosyltransferase herein(e.g., refer to Innis et al., ibid.).

The amino acid substitutions in this method can be any of those one ormore substitutions as disclosed herein. Essentially any non-nativeglucosyltransferase as presently disclosed can be encoded by apolynucleotide as prepared by this method, for instance.

Non-limiting examples of compositions and methods disclosed hereininclude:

1. A non-native glucosyltransferase comprising at least one amino acidsubstitution at a position corresponding with amino acid residueLeu-513, Pro-550, Ser-553, Asn-557, Asn-558, Trp-571, Asn-573, Asp-575,Lys-578, Asn-581, Thr-585, or Gln-616 of SEQ ID NO:62, wherein thenon-native glucosyltransferase synthesizes insoluble alpha-glucancomprising alpha-1,3-glycosidic linkages, and the molecular weight ofthe insoluble alpha-glucan is lower than the molecular weight ofinsoluble alpha-glucan synthesized by a second glucosyltransferase thatonly differs from the non-native glucosyltransferase at the substitutionposition(s).2. The non-native glucosyltransferase of embodiment 1, comprising atleast one amino acid substitution at a position corresponding with aminoacid residue Leu-513, Ser-553, Asn-573, Asp-575, Lys-578, or Gln-616 ofSEQ ID NO:62.3. The non-native glucosyltransferase of embodiment 1 or 2, wherein: (i)the amino acid substitution at the position corresponding with aminoacid residue Leu-513 is with an Ala, Cys, Asp, Glu, Phe, Gly, His, Ile,Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr residue; (ii)the amino acid substitution at the position corresponding with aminoacid residue Pro-550 is with a Leu, Val, or Ile residue; (iii) the aminoacid substitution at the position corresponding with amino acid residueSer-553 is with an Ala, Cys, Glu, Phe, His, Ile, Met, Asn, Arg, Thr,Val, or Tyr residue; (iv) the amino acid substitution at the positioncorresponding with amino acid residue Asn-557 is with a Glu, Gln, Ile,Thr, Asp, Asn, Leu, Val, or Ser residue; (v) the amino acid substitutionat the position corresponding with amino acid residue Asn-558 is with anAsp or Glu residue; (vi) the amino acid substitution at the positioncorresponding with amino acid residue Trp-571 is with a Val, Asp, Cys,Ile, Leu, Glu, or Ser residue; (vii) the amino acid substitution at theposition corresponding with amino acid residue Asn-573 is with an Ala,Asp, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Thr, Val, or Trp residue;(viii) the amino acid substitution at the position corresponding withamino acid residue Asp-575 is with an Ala, Cys, Glu, Phe, Gly, His, Ile,Lys, Leu, Met, Asn, Pro, Arg, Ser, Val, Trp, or Tyr residue; (ix) theamino acid substitution at the position corresponding with amino acidresidue Lys-578 is with an Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Leu,Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr residue; (x) theamino acid substitution at the position corresponding with amino acidresidue Asn-581 is with a Pro or Gly residue; (xi) the amino acidsubstitution at the position corresponding with amino acid residueThr-585 is with a Pro or Gly residue; and/or (xii) the amino acidsubstitution at the position corresponding with amino acid residueGln-616 is with an Ala, Cys, Asp, Glu, Gly, His, Ile, Lys, Leu, Met,Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr residue.4. The non-native glucosyltransferase of embodiment 1, 2, or 3,comprising two or more amino acid substitutions, wherein at least one ofthe substitutions is at a position corresponding with amino acid residueLeu-513, Pro-550, Ser-553, Asn-557, Asn-558, Trp-571, Asn-573, Asp-575,Lys-578, Asn-581, Thr-585, or Gln-616 of SEQ ID NO:62.5. The non-native glucosyltransferase of embodiment 4, comprising atleast one amino acid substitution at a position corresponding with aminoacid residue Pro-550, Asn-557, Asn-558, Asn-581, or Thr-585 of SEQ IDNO:62; optionally wherein: (i) the amino acid substitution at theposition corresponding with amino acid residue Pro-550 is with a Leu,Val, or Ile residue; (ii) the amino acid substitution at the positioncorresponding with amino acid residue Asn-557 is with a Glu, Gln, Ile,Thr, Asp, Asn, Leu, Val, or Ser residue; (iii) the amino acidsubstitution at the position corresponding with amino acid residueAsn-558 is with an Asp or Glu residue; (iv) the amino acid substitutionat the position corresponding with amino acid residue Asn-581 is with aPro or Gly residue; and/or (v) the amino acid substitution at theposition corresponding with amino acid residue Thr-585 is with a Pro orGly residue.6. The non-native glucosyltransferase of embodiment 1, 2, 3, 4, or 5,wherein the insoluble alpha-glucan comprises at least about 50%alpha-1,3 glycosidic linkages.7. The non-native glucosyltransferase of embodiment 6, wherein theinsoluble alpha-glucan comprises at least about 90% alpha-1,3 glycosidiclinkages.8. The non-native glucosyltransferase of embodiment 1, 2, 3, 4, 5, 6, or7, wherein the insoluble alpha-glucan has a weight average degree ofpolymerization (DPw) of less than about 300.9. The non-native glucosyltransferase of embodiment 8, wherein theinsoluble alpha-glucan has a DPw of less than about 150.10. The non-native glucosyltransferase of embodiment 9, wherein theinsoluble alpha-glucan has a DPw of less than about 75.11. The non-native glucosyltransferase of embodiment 6, 7, 8, 9, or 10,comprising a catalytic domain that is at least about 90% identical toSEQ ID NO:66 (residues 55-960 of SEQ ID NO:4), residues 54-957 of SEQ IDNO:65, residues 55-960 of SEQ ID NO:30, residues 55-960 of SEQ ID NO:28,or residues 55-960 of SEQ ID NO:20.12. The non-native glucosyltransferase of embodiment 11, comprising anamino acid sequence that is at least about 90% identical to SEQ ID NO:4,SEQ ID NO:65, SEQ ID NO:30, SEQ ID NO:28, or SEQ ID NO:20.13. The non-native glucosyltransferase of embodiment 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, or 12, wherein the molecular weight of the insolublealpha-glucan is at least about 10% lower than the molecular weight ofinsoluble alpha-glucan synthesized by the second glucosyltransferase.14. A polynucleotide comprising a nucleotide sequence encoding anon-native glucosyltransferase according to any one of embodiments 1-13,optionally wherein one or more regulatory sequences are operably linkedto the nucleotide sequence, and preferably wherein the one or moreregulatory sequences include a promoter sequence.15. A reaction composition comprising water, sucrose, and a non-nativeglucosyltransferase according to any one of embodiments 1-13.16. A method of producing insoluble alpha-glucan comprisingalpha-1,3-glycosidic linkages, the method comprising: (a) contacting atleast water, sucrose, and a non-native glucosyltransferase enzymeaccording to any one of embodiments 1-13, whereby insoluble alpha-glucancomprising alpha-1,3-glycosidic linkages is produced; and (b)optionally, isolating the insoluble alpha-glucan produced in step (a).17. A method of preparing a polynucleotide sequence encoding anon-native glucosyltransferase (e.g., of any one of embodiments 1-13),the method comprising: (a) identifying a polynucleotide sequenceencoding a parent glucosyltransferase that (i) comprises an amino acidsequence that is at least about 40% identical to SEQ ID NO:4 or SEQ IDNO:66 (positions 55-960 of SEQ ID NO:4), and (ii) synthesizes insolublealpha-glucan comprising alpha-1,3-glycosidic linkages; and (b) modifyingthe polynucleotide sequence identified in step (a) to substitute atleast one amino acid of the parent glucosyltransferase at a positioncorresponding with amino acid residue Leu-513, Pro-550, Ser-553,Asn-557, Asn-558, Trp-571, Asn-573, Asp-575, Lys-578, Asn-581, Thr-585,or Gln-616 of SEQ ID NO:62, thereby providing a polynucleotide sequenceencoding a non-native glucosyltransferase that synthesizes insolublealpha-glucan comprising alpha-1,3-glycosidic linkages, wherein themolecular weight of the insoluble alpha-glucan is lower than themolecular weight of insoluble alpha-glucan synthesized by the parentglucosyltransferase.18. The method of embodiment 17, wherein the identifying step isperformed: (a) in silico, (b) with a method comprising a nucleic acidhybridization step, (c) with a method comprising a protein sequencingstep, and/or (d) with a method comprising a protein binding step; and/orwherein the modifying step is performed: (e) in silico, followed bysynthesis of the polynucleotide sequence encoding the non-nativeglucosyltransferase enzyme, or (f) using a physical copy of thepolynucleotide sequence encoding the parent glucosyltransferase.

EXAMPLES

The present disclosure is further exemplified in the following Examples.It should be understood that these Examples, while indicating certainpreferred aspects herein, are given by way of illustration only. Fromthe above discussion and these Examples, one skilled in the art canascertain the essential characteristics of the disclosed embodiments,and without departing from the spirit and scope thereof, can makevarious changes and modifications to adapt the disclosed embodiments tovarious uses and conditions.

Example 1 Analysis of Amino Acid Sites Affecting GlucosyltransferaseAlpha-Glucan Product Molecular Weight

This Example describes screening for glucosyltransferase variants thatproduce alpha-glucan with decreased molecular weight.

The amino acid sequence of the glucosyltransferase used to prepare aminoacid substitutions in this Example was SEQ ID NO:4 (GTF 6855), whichessentially is an N-terminally truncated (signal peptide and variableregion removed) version of the full-length wild type glucosyltransferase(represented by SEQ ID NO:62) from Streptococcus salivarius SK126 (seeTable 1). Substitutions made in SEQ ID NO:4 can be characterized assubstituting for native amino acid residues, as each amino acidresidue/position of SEQ ID NO:4 (apart from the Met-1 residue of SEQ IDNO:4) corresponds accordingly with an amino acid residue/position withinSEQ ID NO:62. In reactions comprising at least sucrose and water, theglucosyltransferase of SEQ ID NO:4 typically produces alpha-glucanhaving about 100% alpha-1,3 linkages and a weight-average degree ofpolymerization (DPw) of 400 or greater (e.g., refer to U.S. Pat. Nos.8,871,474 and 9,169,506, and U.S. Pat. Appl. Publ. No. 2017/0002336,which are incorporated herein by reference). This alpha-glucan product,which is insoluble, can be isolated following enzymatic synthesis viafiltration, for example.

Single mutations were individually introduced into a DNA sequenceencoding SEQ ID NO:4 using a PCR (polymerase chain reaction)-basedmutagenesis protocol (QUIKCHANGE LIGHTNING site-directed mutagenesiskit, Agilent Technologies, Santa Clara, Calif.). For each mutation, aDNA sequence containing the mutation was produced using PCR with a SEQID NO:4-encoding DNA template, a forward primer encoding the mutation,and a shared reverse primer.

Plasmids (pBAD vector-based) for individually expressing various singleamino acid-substituted variants of GTF 6855 (SEQ ID NO:4) were used totransform E. coli strain Bw25113 (ΔilvC), which is a derivative of theF⁻, λ⁻ , E. coli K-12 strain BD792 (CGSC6159) with one additionalknock-out (ΔilvC). LB agar plates with 100 mg/L ampicillin were used toselect transformants. Plasmid DNA from the clones was individuallysequenced to verify that each intended single substitution was presentin a correct manner. To produce GTF 6855 (SEQ ID NO:4) and each singleamino acid-substituted variant thereof, the above E. coli Bw25113(ΔilvC) transformants were individually cultivated overnight at 30° C.in LB media containing 100 mg/L ampicillin and 0.025% L-arabinose. Cellswere then harvested and lysed with BUGBUSTER (EMD Millipore; volume was10% of culture volume) at room temperature for about 1 hour. Lysed cellswere centrifuged to remove cell debris; each resulting supernatant,which contained GTF 6855 (SEQ ID NO:4) or a single aminoacid-substituted variant thereof, was used for glucan polymerizationreactions (below).

GTF 6855 (SEQ ID NO:4) and each single amino acid-substituted variantthereof were individually entered into glucan synthesis reactions withparameters that were the same as, or similar to, the following: vessel,50-mL indented shake flask agitated at 75 rpm; initial pH, 5.7; reactionvolume, 10 mL; sucrose, 400 g/L; GTF, 0.3 mL of culture supernatant(above); KH₂PO4, 5 mM; temperature, 35° C., time, about two days. Thereactions were then heat de-activated at 80° C. for 30 minutes.Insoluble glucan polymers produced in the reactions were individuallyharvested, water-washed, and analyzed for molecular size via a standardsize exclusion chromatography (SEC) approach. Table 3 (below) providesthe DPw of each insoluble glucan product.

TABLE 3 DPw of Insoluble Alpha-1,3-Glucan Produced by GTF 6855 (SEQ IDNO: 4) and Single Amino Acid-Substituted Variants thereof GTF DPw GTFDPw GTF DPw 6855^(a) 350 S553A 127 N573A 123 L513A^(b) 194 S553C 125N573A 125 L513C 119 S553C 126 N573D 108 L513C 159 S553E 105 N573D 134L513D 147 S553E 122 N573G 126 L513D 640 S553F^(c) N573G 120 L513E 129S553F^(c) N573H 148 L513E 130 S553H^(c) N573I^(c) L513F 171 S553H^(c)N573K 145 L513G 138 S553I 79 N573K 148 L513H 153 S553I 97 N573L^(c)L513H 175 S553M 129 N573M^(c) L513I 186 S553M 140 N573N 222 L513K 143S553N 77 N573P 100 L513K 160 S553N 69 N573T 102 L513M 183 S553R 63 N573T109 L513M 210 S553T 226 N573V 91 L513N^(c) S553T 124 N573W 249 L513N 372S553V 86 N573W 237 L513P 173 S553Y 110 L513Q 138 S553Y 52 L513Q 152L513R 134 L513R 141 L513S 138 L513S 152 L513T 146 L513V 175 L513V^(c)L513W 146 L513W 171 L513Y 156 6855^(a) 350 K578A 110 Q616A 175 D575A^(b)74 K578A 113 Q616A 440 D575A 199 K578C 132 Q616C 81 D575C 97 K578D 156Q616D 115 D575C^(c) K578E 95 Q616E 50 D575C 94 K578E^(c) Q616G 66 D575E90 K578F 103 Q616G^(c) D575E 88 K578G 113 Q616H 61 D575F 74 K578G 103Q616I 82 D575G 90 K578H 212 Q616K 58 D575G 89 K578H 187 Q616K 59 D575H70 K578I 179 Q616L 61 D575H 134 K578L 177 Q616L 62 D575I 76 K578M 135Q616M 164 D575I 98 K578M 141 Q616N 269 D575K 52 K578N 185 Q616N 211D575K 95 K578P 126 Q616P 75 D575L 74 K578P 128 Q616P 78 D575L^(c) K578Q111 Q616R 103 D575M 66 K578R 214 Q616R 167 D575M 72 K578R 294 Q616S 72D575N 90 K578S 105 Q616T 79 D575N 191 K578S 105 Q616V 88 D575N^(c) K578T131 Q616V 97 D575P 50 K578T 157 Q616W 60 D575R 65 K578V 146 Q616W 101D575R 71 K578V 145 Q616Y 65 D575S 104 K578W 106 D575S 96 K578W 122 D575V54 K578Y 145 D575W 69 D575W 167 D575Y 124 D575Y 69 ^(a)GTF 6855, SEQ IDNO: 4. The DPw of insoluble alpha-1,3-glucan produced by GTF 6855averaged at about 350. ^(b)Each listed GTF with a substitution is aversion of GTF 6855 comprising a substitution at a respective position,where the position number is in correspondence with the residuenumbering of SEQ ID NO: 62. The wild type residue is listed first(before residue position number) and the substituting residue is listedsecond (after the residue position number) (this “wild typeresidue-position number-variant residue” annotation format appliesthroughout the present disclosure). ^(c)Insoluble alpha-1,3-glucan notproduced or detected.

Based on the data in Table 3, it is apparent that certain amino acidsubstitutions in GTF 6855 (SEQ ID NO:4) result in enzymes that produceinsoluble alpha-1,3-glucan with decreased molecular weight, as comparedto the molecular weight of insoluble alpha-1,3-glucan produced byunmodified GTF 6855 (SEQ ID NO:4). With regard to those enzymes in Table3 that produced insoluble glucan, all but four amino acid substitutionsresulted in decreases in molecular weight by at least about 23%; wellover two-thirds of the amino acid substitutions listed in Table 3resulted in molecular weight decreases of 50% or more. Decreases inmolecular weight of up to about 86% (D575P, Q616E) were observed, forexample.

One or more substitutions at any the foregoing sites in this Example(positions corresponding to Leu-513, Ser-553, Asn-573, Asp-575, Lys-578,or Gln-616 of SEQ ID NO:62) are expected to allow for production ofinsoluble alpha-1,3-glucan with a DPw significantly lower than the DPwof insoluble alpha-1,3-glucan produced by a parent non-substitutedglucosyltransferase.

Example 2 Analysis of the Effects of Amino Acid SubstitutionCombinations on Insoluble Alpha-Glucan Synthesis by Glucosyltransferase

This Example describes the effects of introducing multiple amino acidsubstitutions to a glucosyltransferase and determining their effect onits insoluble alpha-glucan synthesis function. This analysis indicates,for example, that amino acid substitutions identified above to decreaseinsoluble alpha-glucan product molecular weight can be included insubstitution combinations that likewise impart this molecular weighteffect.

Briefly, certain combinations of amino acid substitutions were made toSEQ ID NO:4 (GTF 6855, see Table 1 and Example 1 for description of thisglucosyltransferase) by site-directed mutagenesis. For each combination,the QUIKCHANGE LIGHTNING site-directed mutagenesis kit was used in asuccessive manner to introduce each substitution to a SEQ IDNO:4-encoding sequence. Each combination of substitutions is listed inTable 4 below. The sequences encoding each modified glucosyltransferasewere individually sequenced to confirm the intended changes. Eachamino-acid-modified glucosyltransferase was then expressed and preparedaccording to Example 1.

Glucan synthesis reactions were conducted with each modifiedglucosyltransferase using parameters that were the same as, or similarto, the ones used in Example 1, followed by heat de-activation of eachreaction at 80° C. for 30 minutes. Insoluble glucan polymers produced inthe reactions were individually harvested, water-washed, and analyzedfor molecular size via a standard SEC approach. Table 4 (below) providesthe DPw of each insoluble alpha-1,3-glucan product.

TABLE 4 DPw of Insoluble Alpha-1,3-Glucan Produced by Multiple AminoAcid-Substituted Variants of GTF 6855 (SEQ ID NO: 4) GTF^(a) DPW P550LN557I N581P 12 L535P S553C N558D D575V T585P K697R 12 P550V S553R N581PT585P 12 P550L S553F N581P 12 P550V N557E T585P 12 P550L N557E D575VT585P 13 L538P P550L S553Y 13 P544L P550V S553C N573I T585P S589G 13P550V G576D T585P 13 P550L N558D T585P T679I 13 P550L N557E T585P S589G14 P550L N557E T569L N581P 14 P550L N557E T569L T585P 14 P550V S553TN558D T585P G730D 14 E577G P550L N557I T569L N573I 14 P550L S553C D575AT585P S589G 14 S553R N573V K578N S631G T660A 14 P550V S553R W571V G576D14 P550V N557E K578D T585P 14 P550V N558D N573P T585P 14 P550L N558DW571V N581P K593E 14 P550V S553E N581P 15 P550L N573I T585P W725R 15P550L N557I N573P 15 N557E N573V N581P 15 P550L N557I G576D Q643L 15P550V S553N T585P V586G S710G 15 P550V S553C D575A T585P 15 S553R N573VK578N S631G T660A 15 P550L S553K D575A Y580H 16 P550V D575A T585P S589GK713E 16 P550V S553N N573I Y693C 16 P544L P550L N557E N573I T585P 16P550L N558D W571V N581P T585P 16 S504G P550V N557Q N581P 16 P550L S553RD575A 16 P550V N558D W571D D575A T585P 16 P544L P550V N557Q N581P 17P550V S553K T585P 17 P550V S553N T585P 17 P550L T569L N573I 17 P550LN558D D575V 17 L537P P550L N558D N573I 17 P550L S553C W571C G576D T585P17 P550L N557Q W571C G576D T585P 17 P550L N558D W571V N581P 17 A542VP550V N558D W571V T585P 17 P550V N558D W571D G576D 18 P550V S553N N573I18 P550V N557Q D575V A669T 18 P550V N581P I636T 18 P550V N557E N581P 18P550V N573I T585P 18 P550L S553K N558D K578R Y700N 19 P550V S553T N558DW571V 19 P514L P550V N557Q T585P D602N 20 P550L N557I T569A G576D 20P550V N557T N558D W571D 21 P550L N557E D575A T585P 21 I545V P550V N557QT585P D638N 21 P544L P550V N557I T585P 22 Y518C P550V N581P T585P 22P550L N557E D575A 22 ^(a)Each listed GTF is a version of GTF 6855 (SEQID NO: 4) comprising substitutions at respective positions, where eachposition number is in correspondence with the residue numbering of SEQID NO: 62.

Based on the data in Table 4, it is apparent that introduction ofmultiple amino acid substitutions to GTF 6855 (SEQ ID NO:4)—somecombinations of which include substitutions shown in Example 1 todecrease molecular weight—can be employed in efforts to produce lowermolecular weight insoluble alpha-1,3-glucan. For example, compare theseDPw values (Table 4) to the -350 DPw of insoluble alpha-1,3-glucanproduced by GTF 6855 (SEQ ID NO:4) without substitutions (Table 3).

It is apparent that a glucosyltransferase with multiple substitutions,including for example those at positions corresponding to (i) Pro-550,Asn-557, Asn-558, Asn-581, and/or Thr-585 of SEQ ID NO:62, and/or (ii)Leu-513, Ser-553, Asn-573, Asp-575, Lys-578, and/or Gln-616 of SEQ IDNO:62, can produce insoluble alpha-1,3-glucan with decreased molecularweight.

Example 3 Reactions Containing Dextran with 18.6% Alpha-1,2 Branches andGlucosyltransferase that Produces Low Molecular Weight Alpha-1,3-Glucan

This Example describes synthesis of alpha-1,3-glucan inglucosyltransferase reactions containing a dextran primer that waspreviously modified to have 18.6% alpha-1,2 branches. Theglucosyltransferase enzyme used in these reactions was modified (asdisclosed in above Examples) to produce alpha-1,3-glucan of lowermolecular weight compared to glucan product made by the enzyme'sunmodified counterpart. Graft copolymers were synthesized comprising adextran backbone and alpha-1,3-glucan arms.

Alpha-1,2-branched dextran was produced to prime glucosyltransferasereactions for alpha-1,3-glucan synthesis. Preparation of this primer wasperformed essentially as described in International Patent. Appl. Publ.No. WO2017/091533 and U.S. Patent Appl. Publ. No. 2018/0282385, whichare both incorporated herein by reference. Briefly, linear dextran(i.e., polysaccharide with 100% alpha-1,6 glycosidic linkages) ofmolecular weight ˜14 kDa (DPw of ˜89) was produced usingglucosyltransferase GTF8117. This dextran was then modified to have18.6% alpha-1,2-branches using alpha-1,2-branching enzyme GTFJ18T1. Theresulting dextran, which had a DPw of about 104 (˜17 kDa) and 18.6%alpha-1,2-branches, is referred to herein as P5 primer.

P5 primer was then included in alpha-1,3-glucan synthesis reactionscomprising a glucosyltransferase to produce graft copolymers comprisinga dextran backbone and alpha-1,3-glucan arms. The glucosyltransferaseused in these reactions is a non-native one as described in Example 2above (Table 4). Enzyme preparation in the present Example was conductedas follows. Briefly, KOBW-3a cells heterologously expressing theglucosyltransferase were grown in LB medium containing 100 ng/mLampicillin and 0.025% L-arabinose at 30° C. overnight. Cells werepelleted by centrifugation and then lysed with a 10% volume ofBugBuster® Protein Extraction Reagent (EMD Millipore) at roomtemperature for at least 1 hour; cell debris was removed bycentrifugation, after which clear cell lysate was collected for testing.

Thirteen separate 4.0-mL alpha-1,3-glucan synthesis reactions wereperformed in glass 50-mL indented flasks. Each reaction comprised water,sucrose (roughly 20, 50, or 200 g/L), phosphate buffer (5 mM, pH 5.7),optionally P5 primer (roughly 0.2, 1.0, or 5.0 g/L), andglucosyltransferase (above). The reactions were prepared by mixing anappropriate amount of P5 primer (using 50 g/L stock solution), 0.2 mL ofcell lysate (containing the glucosyltransferase) and correspondingbuffer. Each of these preparations and a sucrose stock solution (400g/L) was warmed at 35° C. for at least 30 minutes. An appropriate volumeof sucrose solution was then added to each preparation to initiatealpha-1,3-glucan synthesis. The reactions were carried out at 35° C.with shaking (10 rpm) for about 60 hours. Following this incubation, theentire contents of the reactions were individually transferred to 15-mLcentrifuge tubes, which were placed in an 85° C. oven for about 30minutes to deactivate the glucosyltransferase thereby terminating thereactions. Approximately 300-500 mg of wet cake produced in eachreaction was analyzed by SEC to determine the molecular weight and DP ofthe insoluble polymer products. Table 5 provides the results of theseanalyses.

TABLE 5 Analysis of Alpha-1,3-Glucan Produced in GlucosyltransferaseReactions Containing P5 Primer Reaction Primer^(a) SucroseAlpha-1,3-Glucan Product Profile (g/L) (g/L) Polymer Type DPw Mw MpMw/Mn 0.0 20 non-grafted 58 9439 9948 1.22 0.2 grafted 625 101191 977261.33 1.0 grafted 402 65163 62668 1.32 5.0 grafted 245 39628 35874 1.210.0 50 non-grafted 52 8363 8117 1.25 0.2 grafted 680 110189 95028 1.361.0 grafted 425 68878 68877 1.28 5.0 grafted 251 40718 36926 1.22 0.0200 non-grafted 41 6690 5945 1.22 0.2 grafted 517 83679 88410 1.3 1.0grafted 374 60530 59285 1.24 5.0 grafted 236 38264 35119 1.2 ^(a)PrimerP5 is DPw 104 (Mw = 16869 Da) (Mw/Mn = 1.48).

The size of individual alpha-1,3-glucan arms grafted onto primer in theglucan product of each primer-containing reaction is contemplated to bevery similar to the size of alpha-1,3-glucan produced in controlreactions that did not contain added primer; the control reactionproducts comprised alpha-1,3-glucan homopolymer. That said, it seemsnotable that, as the concentration of primer was increased in each setof reaction series (20, 50, or 200 g/L sucrose), graft copolymer productDPw decreased significantly.

Thus, graft copolymers were synthesized comprising a dextran backboneand alpha-1,3-glucan arms using a non-native glucosyltransferase of theinstant disclosure. Primer was demonstrably consumed and incorporatedinto graft copolymer products. While alpha-1,2-branched dextran was usedas primer in this Example, alpha-1,3-glucan synthesis was also observedwhen using dextran (as described above) that had not been modified withalpha-1,2 branches (data not shown).

Example 4 Reactions Containing Dextran with 9.7% Alpha-1,2 Branches andGlucosyltransferase that Produces Low Molecular Weight Alpha-1,3-Glucan

This Example describes synthesis of alpha-1,3-glucan inglucosyltransferase reactions containing a dextran primer that waspreviously modified to have 9.7% alpha-1,2 branches. Theglucosyltransferase enzyme used in these reactions was modified (asdisclosed in above Examples) to produce alpha-1,3-glucan of lowermolecular weight compared to glucan product made by the enzyme'sunmodified counterpart. Graft copolymers were synthesized comprising adextran backbone and alpha-1,3-glucan arms.

Alpha-1,2-branched dextran was produced to prime glucosyltransferasereactions for alpha-1,3-glucan synthesis. Preparation of this primer wasperformed essentially as described in International Patent. Appl. Publ.No. WO2017/091533 and U.S. Patent Appl. Publ. No. 2018/0282385. Briefly,linear dextran (i.e., polysaccharide with 100% alpha-1,6 glycosidiclinkages) of molecular weight ˜40 kDa (DPw of ˜250) was produced usingglucosyltransferase GTF6831. This dextran was then modified to have 9.7%alpha-1,2-branches using alpha-1,2-branching enzyme GTFJ18T1. Theresulting alpha-1,2-branched dextran, which had a DPw of about 271 (˜44kDa), is referred to herein as P6 primer.

P6 primer was then included in alpha-1,3-glucan synthesis reactionscomprising a glucosyltransferase to produce graft copolymers comprisinga dextran backbone and alpha-1,3-glucan arms. The glucosyltransferaseused in these reactions was the same as used and prepared in Example 3.

Thirteen separate 4.0-mL alpha-1,3-glucan synthesis reactions wereperformed in glass 50-mL indented flasks. Each reaction comprised water,sucrose (roughly 20, 50, or 200 g/L), phosphate buffer (5 mM, pH 5.7),optionally P6 primer (roughly 0.2, 1.0, or 5.0 g/L), andglucosyltransferase (above). The reactions were prepared by mixing anappropriate amount of P6 primer (using 50 g/L stock solution), 0.2 mL ofcell lysate (containing the glucosyltransferase) and correspondingbuffer. Each of these preparations and a sucrose stock solution (400g/L) was warmed at 35° C. for at least 30 minutes. An appropriate volumeof sucrose solution was then added to each preparation to initiatealpha-1,3-glucan synthesis. The reactions were carried out at 35° C.with shaking (10 rpm) for about 60 hours. Following this incubation, theentire contents of the reactions were individually transferred to 15-mLcentrifuge tubes, which were placed in an 85° C. oven for about 30minutes to deactivate the glucosyltransferase thereby terminating thereactions. Approximately 300-500 mg of wet cake produced in eachreaction was analyzed by SEC to determine the molecular weight and DP ofthe insoluble polymer products. Table 6 provides the results of theseanalyses.

TABLE 6 Analysis of Alpha-1,3-Glucan Produced in GlucosyltransferaseReactions Containing P6 Primer Reaction Primer^(a) SucroseAlpha-1,3-Glucan Product Profile (g/L) (g/L) Polymer Type DPw Mw MpMw/Mn 0.0 20 non-grafted 58 9439 9948 1.22 0.2 grafted 1224 198295222340 1.55 1.0 grafted 1023 167308 177954 1.7 5.0 grafted 769 12464695437 1.6 0.0 50 non-grafted 52 8363 8117 1.25 0.2 grafted 1211 196134224349 1.59 1.0 grafted 1085 175800 187403 1.58 5.0 grafted 768 124454112146 1.64 0.0 200 non-grafted 41 6690 5945 1.22 0.2 grafted 993 160848206992 1.58 1.0 grafted 1060 171788 184475 1.45 5.0 grafted 749 121391114253 1.61 ^(a)Primer P6 is DPw 271 (Mw = 43958 Da) (Mw/Mn = 1.19).

The size of individual alpha-1,3-glucan arms grafted onto primer in theglucan product of each primer-containing reaction is contemplated to bevery similar to the size of alpha-1,3-glucan produced in controlreactions that did not contain added primer; the control reactionproducts comprised alpha-1,3-glucan homopolymer. That said, it seemsnotable that generally, as the concentration of primer was increased ineach set of reaction series (20, 50, or 200 g/L sucrose), graftcopolymer product DPw decreased significantly. Thus, similarly toExample 3, graft copolymers were synthesized comprising a dextranbackbone and alpha-1,3-glucan arms using a non-nativeglucosyltransferase of the instant disclosure. Primer was demonstrablyconsumed and incorporated into graft copolymer products.

What is claimed is:
 1. A non-native glucosyltransferase comprising anamino acid sequence that is at least 90% identical to SEQ ID NO:66,wherein said amino acid sequence has at least one amino acidsubstitution at a position corresponding with amino acid residueLeu-282, Pro-319, Ser-322, Asn-326, Asn-327, Trp-340, Asn-342, Asp-344,Lys-347, Asn-350, Thr-354, or Gln-385 of SEQ ID NO:66, wherein thenon-native glucosyltransferase uses sucrose to synthesize insolublealpha-glucan comprising at least 50% alpha-1,3-glycosidic linkages, andthe molecular weight of said insoluble alpha-glucan is at least 10%lower than the molecular weight of insoluble alpha-glucan synthesized bya second glucosyltransferase that only differs from the non-nativeglucosyltransferase at the substitution position(s).
 2. The non-nativeglucosyltransferase of claim 1, comprising at least one amino acidsubstitution at a position corresponding with amino acid residueLeu-282, Ser-322, Asn-342, Asp-344, Lys-347, or Gln-385 of SEQ ID NO:66.3. The non-native glucosyltransferase of claim 1, wherein: (i) the aminoacid substitution at the position corresponding with amino acid residueLeu-282 is with an Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Lys, Met,Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr residue; (ii) the aminoacid substitution at the position corresponding with amino acid residuePro-319 is with a Leu, Val, or Ile residue; (iii) the amino acidsubstitution at the position corresponding with amino acid residueSer-322 is with an Ala, Cys, Glu, Phe, His, Ile, Met, Asn, Arg, Thr,Val, or Tyr residue; (iv) the amino acid substitution at the positioncorresponding with amino acid residue Asn-326 is with a Glu, Gln, Ile,Thr, Asp, Asn, Leu, Val, or Ser residue; (v) the amino acid substitutionat the position corresponding with amino acid residue Asn-327 is with anAsp or Glu residue; (vi) the amino acid substitution at the positioncorresponding with amino acid residue Trp-340 is with a Val, Asp, Cys,Ile, Leu, Glu, or Ser residue; (vii) the amino acid substitution at theposition corresponding with amino acid residue Asn-342 is with an Ala,Asp, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Thr, Val, or Trp residue;(viii) the amino acid substitution at the position corresponding withamino acid residue Asp-344 is with an Ala, Cys, Glu, Phe, Gly, His, Ile,Lys, Leu, Met, Asn, Pro, Arg, Ser, Val, Trp, or Tyr residue; (ix) theamino acid substitution at the position corresponding with amino acidresidue Lys-347 is with an Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Leu,Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr residue; (x) theamino acid substitution at the position corresponding with amino acidresidue Asn-350 is with a Pro or Gly residue; (xi) the amino acidsubstitution at the position corresponding with amino acid residueThr-354 is with a Pro or Gly residue; and/or (xii) the amino acidsubstitution at the position corresponding with amino acid residueGln-385 is with an Ala, Cys, Asp, Glu, Gly, His, Ile, Lys, Leu, Met,Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr residue.
 4. The non-nativeglucosyltransferase of claim 1, comprising at least one amino acidsubstitution at a position corresponding with amino acid residuePro-319, Asn-326, Asn-327, Asn-350, or Thr-354 of SEQ ID NO:66.
 5. Thenon-native glucosyltransferase of claim 1, wherein the insolublealpha-glucan comprises at least about 80% alpha-1,3 glycosidic linkages.6. The non-native glucosyltransferase of claim 5, wherein the insolublealpha-glucan comprises at least about 90% alpha-1,3 glycosidic linkages.7. The non-native glucosyltransferase of claim 1, wherein the insolublealpha-glucan has a weight average degree of polymerization (DPw) of lessthan about
 300. 8. The non-native glucosyltransferase of claim 7,wherein the insoluble alpha-glucan has a DPw of less than about
 150. 9.The non-native glucosyltransferase of claim 8, wherein the insolublealpha-glucan has a DPw of less than about
 75. 10. The non-nativeglucosyltransferase of claim 1, wherein the molecular weight of saidinsoluble alpha-glucan is at least 25% lower than the molecular weightof insoluble alpha-glucan synthesized by said secondglucosyltransferase.
 11. A reaction composition comprising water,sucrose, and a non-native glucosyltransferase according to claim
 1. 12.A method of producing insoluble alpha-glucan comprising 1,3-glycosidiclinkages, the method comprising: contacting at least water, sucrose, anda non-native glucosyltransferase enzyme according to claim 1, wherebyinsoluble alpha-glucan comprising 1,3-glycosidic linkages is produced.13. The method of claim 12, further comprising a step of isolating theinsoluble alpha-glucan.
 14. The method of claim 12, wherein theinsoluble alpha-glucan comprises at least about 80% alpha-1,3 glycosidiclinkages.
 15. The method of claim 12, wherein the insoluble alpha-glucanhas a weight average degree of polymerization (DPw) of less than about300.
 16. The method of claim 12, wherein the non-nativeglucosyltransferase comprises an amino acid sequence that is at leastabout 95% identical to SEQ ID NO:66.
 17. The non-nativeglucosyltransferase of claim 2, wherein: (i) the amino acid substitutionat the position corresponding with amino acid residue Leu-282 is with anAla, Cys, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg,Ser, Thr, Val, Trp, or Tyr residue; (ii) the amino acid substitution atthe position corresponding with amino acid residue Ser-322 is with anAla, Cys, Glu, Phe, His, Ile, Met, Asn, Arg, Thr, Val, or Tyr residue;(iii) the amino acid substitution at the position corresponding withamino acid residue Asn-342 is with an Ala, Asp, Gly, His, Ile, Lys, Leu,Met, Asn, Pro, Thr, Val, or Trp residue; (iv) the amino acidsubstitution at the position corresponding with amino acid residueAsp-344 is with an Ala, Cys, Glu, Phe, Gly, His, Ile, Lys, Leu, Met,Asn, Pro, Arg, Ser, Val, Trp, or Tyr residue; (v) the amino acidsubstitution at the position corresponding with amino acid residueLys-347 is with an Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Leu, Met,Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr residue; and/or (vi) theamino acid substitution at the position corresponding with amino acidresidue Gln-385 is with an Ala, Cys, Asp, Glu, Gly, His, Ile, Lys, Leu,Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr residue.
 18. Thenon-native glucosyltransferase of claim 4, wherein: (i) the amino acidsubstitution at the position corresponding with amino acid residuePro-319 is with a Leu, Val, or Ile residue; (ii) the amino acidsubstitution at the position corresponding with amino acid residueAsn-326 is with a Glu, Gln, Ile, Thr, Asp, Asn, Leu, Val, or Serresidue; (iii) the amino acid substitution at the position correspondingwith amino acid residue Asn-327 is with an Asp or Glu residue; (iv) theamino acid substitution at the position corresponding with amino acidresidue Asn-350 is with a Pro or Gly residue; and/or (v) the amino acidsubstitution at the position corresponding with amino acid residueThr-354 is with a Pro or Gly residue.
 19. The non-nativeglucosyltransferase of claim 1, wherein the non-nativeglucosyltransferase comprises an amino acid sequence that is at leastabout 95% identical to SEQ ID NO:66.